Phage spleen

Krebber, A., S. Bornhauser, J. Burmester, A. Honegger, J. Willuda, H.R. Bosshard, and A. Pluckthun, Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. J Immunol Methods, 1997. 201(1) 35-55. 

Glucosylated oligonucleotides obtained from T4 phage DNA by acid DNase digestion are resistant to spleen exonuclease (28). It has been reported that acetylation of the 2 -OH groups of tRNA completely inhibits the action of the enzyme, whereas venom exonuclease is not affected (29). The naturally occurring methylation of sugars and bases in tRNA does not seem to hinder the action of spleen exonuclease. 

This discussion has focused on the desire to create a one-pot naive phage antibody library. However, phage display has also been used very effectively to isolate binding antibodies when libraries are made from immunized experimental animals [32], or from naturally immunized humans [7, 30] in these cases the V-gene source being either spleen or peripheral-blood lymphocytes. 

The phage S5 tem replaces all steps after immunization and isolation of spleen cells with simple, rapid procedures utilizing DNA manipulation and bacteria. The time required from isolation of spleen cells to selected stable clones is re-... 

In most cases, the spleen is the best choice for antigen-specific lymphocyte isolation. The immunization regime used to immunize mice is identical in every respect to that used for conventional hybridoma production. In particular about ten days after each injection, test bleeds should be carried out to test for the presence of specific antibodies and only animals giving the best antiserum should be selected. Do not expect to recover high affinity antibodies from phage antibody libraries that are not foimd in the test serum. Three to five days before harvesting the spleen for RNA extraction, the immunized mouse is given a final boost. This is best achieved by an intravenous injection done concurrently with an intraperitoneal injectiou... 

---