Hi. I want to generate antibodies by the phage display technique but I´m quite confused about the generation of these proteins when there is pre-made libraries available.
Do I need to immunize an animal to isolate the genes of light and heavy chains?
My problem is that I don´t understand how I can use the pre-made libraries (I don´t know if I have to synthesize the genes besides the primers or something like this?)
Thanks a lot.
If you have a pre-made library, you can just start selecting. A library consist of on the order of billions different antibody scFv or fab genes, and you just select the ones that actually recognize your antigen. Pre-made antibody libraries represent a naive antibody repertoire (or its synthetic equivalent) - If the repertoire is diverse enough, there are antibodies against your antigen present, and you just have to enrich them. The alternating panning and amplification steps replace the the B-cell clonal selection system of in-vivo immunization. Typically, the library consist either already of phages containing the antibody genes, or the plasmids which you electroporate into e-coli and package into phages by infection with helper phages.
Hi Cristian
Annemarie has explained completely. You can refer to this protocol for details of the method:
http://www.nature.com/nprot/journal/v9/n3/full/nprot.2014.039.html?foxtrotcallback=true
If you use a pre-made library, ask the provider of this library for the specific protocols. In addition, the nature and quality of your antigen is of vital importance. The protocol cited by Tahereh addresses the added complication of panning against a membrane protein. An important factor is keeping the antigen-independent unspecific retention of phages to a minimum, especially if you choose to increase stringency in off-rate selection steps for high affinity binders: e.g. by prepanning in the absence of antigen to remove phages that bind to the panning surface/magnetic beads independent of the antigen and by alternating between neutravidin and streptavidin in the different panning rounds to get rid of antibodies recognizing the avidin used for immobilization. If first generation binders from a naive library do not reach very high affinity, a round of error-prone PCR followed by off-rate selection may mimic the affinity gain produced by the affinity maturation achieved by somatic hypermutation in the B-cells of an immune library.
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