I have cloned scFV against various target proteins by infecting XL-1B cells containing phagemid pSEX81 with M13 hyperphage (Progen protocol http://www.progen.de/hyperphage.html). The titre obtained was very low and binding with target protein was also weak in ELISA. How can I amplify both the phage titre and binding?
What was the concentration of your phages? The usual phage titer in my lab is 10 13 particles/mL after PEG precipitation. I would need to know whether your titer is more or less in that normal range or not.
Depending on this answer, it would be posible to determine whether this is the reason of your problem or not. Just in case, have these scFvs been tested in the phage display format before?
The titre of phages that I obtain is usually in the range of e10 to e11 pfu/mL. But in one experiment, I found out to be e7 pfu/mL which was very low and it was important to have more phages for that assay.
And as per my knowledge, I have not seen reports of these ScFvs being tested in phage display?
Why pfu? How are you titrating your phages? I titrate phagemid-containing viral particles by infecting E.coli and growing them on amp (if your phagemid has an amp-resistance gene), so I get cfu/mL. My titers are usually higher.
Some molecules are not properly displayed on phages and they lose their functional properties. This could be the case of your scFvs. Do you have a control? I mean a molecule that has been successfully displayed and could be used as a positive control once displayed using your phagemid.
I am facing this main critical issue. I want advice on the suitable control that can be used in my assay. Sequencing analysis showed some issues with the phagemid vector that I have. We are ordering new one, but need to have a control. Kindly suggest.
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