Abstract
A rapid route for the generation of monoclonal antibodies by repertoire cloning is described. The technique circumvents the need for conventional hybridoma production and allows a variety of new approaches in antibody design and selection. The genes encoding the Fd and light chains are accessed by PCR amplification from cDNA and cloned into two separate λ expression vectors. The libraries of λ Fd and λ light chains are then combined to generate a single λ combinatorial vector encoding both chains and capable of generating a Fab fragment. Since the libraries are constructed in bacteriophage λ, in vitro packaging offers a very efficient route for reintroducing the recombinant DNA back into Escherichia coli . The library generated in such a way may be screened by conventional plaque lifts on nitrocellulose filters using labeled antigen.
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