phage display info.

Mick Foley m.foley at latrobe.edu.au
Sun Jun 30 21:22:47 EST 1996


Dear All,

Concerning the use of Pharmacia recombinant phage display system. We 
bought the kit and have used part of it (pCANTAB5E and the primers) to 
construct a scFv library from hyperimmunised mice. Other procedures such 
as mRNA purification etc we stuck with what we already knew, although we 
followed the general stategy outlined in the pharmacia notes. The library 
we made was only small less than 100,000 clones but after four rounds of 
panning we isolated four different antibody sequences. The phage bind to 
the antigen and not to BSA on ELISA and dot blot. Soluble phage were easy 
to produce and were found to bind to the antigen used for immunisation 
and panning. Our feelings are : the vector and primers work well with 
hyperimmunised spleens (let the mouse do the hard work). We may not get 
all the antibody specificities but even if we only get some we still get 
some. In this respect it may actually be more tricky to get scFv from a 
hybridoma since there is only one real sequence you may not have the 
appropriate primers. And there is a problem with abnormal light chain 
expression. In addition if you obtain your scFv after four rounds of 
panning you select not only for binders but also for scFv that produce 
well in bacteria (if it doesn't get to the periplasm it doesn't get on 
the phage thus it doesn't get selected). No such guarantee with the 
hybridoma. We are in the process of making other libraries uing the same 
strategy from immunised mouse spleens.

Anyway I hope our experiences have been of some help to some of you our 
experience has been a positive one but I have heard some different 
stories.

Cheers,

Mick






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