I'm currently using a phage display technology - constructed nanobody library to screen for nanobodies with relatively high affinity, with CV - A10 virus - like particles as the antigen. After three rounds of in - vitro screening with increasing intensity, ELISA characterization was performed. Sequencing of the positive strains detected revealed that this strain had not been transformed with the nanobody sequence (there was a 10% empty - loading rate in the library construction). We performed molecular docking simulations between the G8P protein of M13 phage tail and CV - A10 virus - like particles, and found that they have an amazing possibility to bind. Have we discovered a defect in this display system? We urgently need your help with this issue!!!
Thank you in advance!!!
No, that's something you should be aware of as a phage display user. That's why it's often worth doing pre-clearing before the actual selection. And one of many reasons why no amount of ELISAs will help you predict what happens when you actually purify your antibody fragment and try to form a complex with it.
Apologies for bothering you again. Could you please explain what "pre - clearing" means? I once pre - incubated the library with blocking buffer before screening, then removed components that specifically bind to the proteins in the blocking buffer, but it didn't seem to help with the issue. QAQ
Do you immobilize your target on beads? That's how this is typically done. In that case, pre-clearing means incubating your library with blocked bead with no target immobilized (or if you have some tag on a target it helps also to immobilize just tag).
I roughly got your point! I've not experimented with beads. We block antigens in ELISA tubes. In theory, we also perform a pre-clearing experiment similar to what you described, yet it doesn't significantly help eliminate empty phages. Thanks for your reply again!
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