Altering the fine specificity of an anti-Legionella single chain antibody by a single amino acid insertion.

Antibody engineering provides the potential to clone and manipulate antibody genes to produce fragments with altered specificity. We have produced an anti- Legionella single chain fragment with broader specificity towards Legionella serotypes than the parent monoclonal antibody. Using this relationship between the parent monoclonal and the recombinant antibody derived from it as a model, we attempted to identify those residues responsible for this change in fine specificity. Sequence analysis of this recombinant antibody revealed the deletion of a conserved residue, Asp101, in the CDR-H3 region. Using site-directed mutagenesis, we have created a mutant form of this single chain fragment with an aspartic acid insertion mutation at position 101 of the antibody heavy chain. This mutant scFv demonstrates improved specificity compared to the wild-type recombinant antibody, indicating an important role for Asp101.