Recombinant antibodies are typically scFvs (single chain variable fragments), Fabs, or IgGs created by cloning a scFv or Fab into an IgG framework. Existing hybridomas also can be converted to recombinant antibodies by synthesizing a clone using the hybridoma DNA sequence and expressing the clone, either in scFv, Fab, or IgG formats in bacterial or mammalian cells.
Recombinant antibodies are most frequently developed using phage display methods where binding molecules (candidate antibodies) are selected from a large recombinant antibody library. In this technique, large numbers (>1010) of different antibodies are displayed on the surface of the bacteriophage M13 as a genetic fusion to one of the phage coat proteins, such as Gp3, and are physically linked to their coding DNA which resides within the phage virion.
Traditional phage display involves solid media plating steps that are labor intensive and low-throughput. Specific binders are selected by enrichment of binding phage during cycles of biopanning against the protein of interest and propagation of the binding phage. In order to derive a high affinity antibody, and affinity maturation step is often required. Hence, the cost and development time associated with recombinant antibody technologies has until now provided no incentive or research and diagnostics groups to utilize antibody discovery methods other than hybridomas or classical polyclonal methods.
Since no immunization steps are required, comprehensive phage-antibody libraries permit targeting of antigens in vitro which are known to be toxic or possess low antigenicity in vivo. High affinity phage antibodies have been selected against numerous cellular proteins and small molecules. In addition, the DNA encoding the antibody is accessible in phage display, providing not only the antibody sequence, but facilitating cloning and engineering of the protein in many ways. Recombinant antibodies are frequently utilized in immunoassays as scFvs linked to detection tags such as DDK (Flag®). A scFv recombinant antibody can also be converted other formats such as an IgG or a scFv-alkaline phosphatase conjugate. The scFv and scFv-alkaline phosphatase conjugate formats offer production cost advantages as both can be produced in E. coli.
Common recombinant antibody applications:
- Western blotting
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