IG GENE LIBRARIES CONSTRUCTION:

A quite different approach for generating monoclonal antibodies employs the polymerase chain reaction (PCR) to amplify the DNA encoding antibody heavy chain and light chain Fab fragments from hybridoma cells. A promoter region and EcoRI site are added to the amplified sequences and the resulting constructs are inserted into bacteriophage lambda, yielding separate heavy and light chain libraries. Cleavage with EcoRI and random joining of the heavy and light chain genes yield numerous novel heavy lights constructs. This procedure generates enormous diversity of antibody specificities; clones containing these random combinations of H + L chains can be rapidly screened for those secreting antibody to a particular antigen.

For example, in one study a million clones were screened in just 2 days, with over 100 clones being identified that produced antibody specific for the desired antigen. The technique has the potential of producing an enormous repertoire of antibody specificities without the limitations of antigen priming and hybridoma technology that currently complicate the production of monoclonal antibodies.