Bypassing Hybridoma Technology Using Phage Display

Phage display can be used to bypass hybridoma technology to make scFv or Fab antibodies from immunized mice, rabbits, chickens or humans or from humans who have mounted an immune response as a result of infection or a disease process. Immune lymphocytes are harvested from peripheral blood lymphocytes, spleen or bone marrow and RNA prepared.
scFv or Fab gene repertoires are prepared using PCR and used to construct phage antibody libraries from which antigen specific antibodies are isolated using affinity chromatography.
In the first example, an scFv phage antibody library was created from the V genes of a mouse immunized with the hapten phenyloxazolone. An scFv phage antibody library was created from murine V genes and binding phage antibodies were isolated by selection on an antigen column.
After two rounds of selection, more than 20 unique scFvs were isolated. The Kd of the highest affinity phage antibodies (1.0*10^-8 M) were comparable to the affinities of IgG from hybridomas constructed from mice immunized with the same hapten. Similar panels of scFv have been obtained using phage display and mice immunized with EGF receptor and botulinum neurotoxin type A. This approach has also been used to produce monoclonal chicken and rabbit antibody fragments using species specific primers. Antigen specific antibodies can also be isolated from phage antibody libraries constructed from the V genes of immunized humans or from humans mounting an immune response to an infection or disease process.
Since hybridoma technology has not worked well to generate human hybridomas, this approach is especially useful for the generation of human antibodies where an immune response can be induced or is mounted. Human monoclonal antibody fragments have been isolated from immunized volunteers or infected patients against tetanus toxin, botulinum neurotoxin, HIV- hepatitis B, hepatitis C, respiratory syncytial virus and hemophilus influenza.Autoimmune antibodies have been isolated from patients with SLE and myasthenia gravis, and also other autoimmune diseases. There has been success in isolating antibodies to self-antigens, including tumor antigens, from libraries constructed from patients with disease or after vaccination with tumor antigen.