Cloning of Variable Domains from Mouse Hybridoma by PCR

Some hybridoma cell lines are very low producers, difficult to cultivate, or antibody production is lost upon prolonged culture. In these cases, a recombinant "hybridoma immortalization" can rescue a valuable antibody for further unlimited propagation in E. coli or other recombinant production systems. Further, monoclonal antibodies with promising specificity, but low affinity, can be improved by phage display, or mouse antibodies can be humanised for therapy. This chapter describes sets of thoroughly validated oligonucleotide primers for a PCR procedure to isolate and clone the DNA of antibody Fv fragments (antigen binding region) from mouse and rat hybridoma cell lines. The protocol includes a colony staining assay to identify E. coli clones producing scFv antibody fragments.