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.
McCarthy
B J
BJ
CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia. Barry-McMarthy@amt-sybex.com
Hill
A S
AS
D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000235:genetics*; D000911:Antibodies, Monoclonal / Q000235:genetics*; D000918:Antibody Specificity; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D022801:Complementarity Determining Regions; D017931:DNA Primers / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D016952:Legionella pneumophila / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D017386:Sequence Homology, Amino Acid
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D022801:Complementarity Determining Regions; D017931:DNA Primers; D004274:DNA, Recombinant; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins