Titration & Amplification of Virus
Amplification of virus inoculum for experimental work or protein production
As part of the service provided to make a recombinant virus, we produce a 50 ml high titre virus stock. This is then used to produce further batches of virus inoculum for protein production.
Clients may also send us their virus that they have already made and we can amplify it to high titre. We always test the virus titre of received viruses by plaque assay before proceeding to large-scale amplification of virus inoculum.
We can amplify virus up to 40 L in one batch. To give an idea of the volumes of virus required for protein production the following may be useful:
|Titre of virus||MOI||Cell density||Cell volume||Volume of virus required|
|1 x 108 pfu/ml||10||2 x 106/ml||1,000ml||200ml|
|2 x 108 pfu/ml||10||2 x 106/ml||1,000ml||100ml|
|5 x 107 pfu/ml||10||2 x 106/ml||1,000ml||400ml|
|1 x 108 pfu/ml||10||2 x 106/ml||10,000ml||2,000ml|
High titre virus stocks are produced using Sf9 cells in serum-free medium. After infection, the cells are removed by centrifugation and the virus stored at 4°C in the dark. Aliquots of virus can be prepared for long term storage. The titre is determined by plaque-assay or baculoQUANT.
Titration of Virus Using baculoQUANT™ ALL-IN-ONE qPCR
OET has developed a rapid system based on quantitative PCR (qPCR) for the accurate titration of baculovirus stocks. In this method, the baculovirus DNA is purified from the budded virus using freshly amplified virus inoculum. The purified DNA is added to a qPCR reaction mix and then amplified in an ABI7500 Sequence Detection System. Following amplification, the quantity of virus particles is determined against the known standards that have been titrated by plaque assay. While this method has proven to be as accurate as plaque assay in our experience, it is not recommended for virus stocks older than three months. Spurious results are obtained from old virus stocks as a result of aggregation and degradation of the virus particles. A plaque assay can determine the titre of an old virus stock more accurately, click here to find out more out our plaque assay services.
Our service offers an optimised method for titration of virus, based on the plaque assay, to determine an accurate infectious titre of the recombinant baculovirus stock. Customers need to provide at least 0.5ml of virus inoculum for the baculoQUANT™ ALL-IN-ONE titration method.
The baculoQUANT™ ALL-IN-ONE titration kit is also available to buy from our online shop
Virus titration using plaque assay
Why is it important to know the titre of a baculovirus preparation?
The viral titre allows infection of the cells to be performed at a known multiplicity of infection (MOI). To achieve maximum production of recombinant protein, an optimal MOI is used. Amplification of virus stocks is also best achieved by using a low MOI to infect cells.
What is a plaque assay?
In the plaque assay, cell monolayers are infected with a serial dilution of the virus stock solution and an agarose overlay is used to restrict the flow of virus. Cells that are infected release progeny virus, however, this only allows infection of neighbouring cells. Lysis of the infected cells produces clear regions or plaques. The plaques are then visualised using a dye such as neutral red.
What other method does OET offer to determine baculovirus titres?
We also offer a quantitative PCR method based on the baculoQUANT system to determine highly accurate titres for fresh stocks. See above.
To discuss your needs or to request a quote, please contact us at firstname.lastname@example.org.