Selection of high affinity p-azophenyarsonate Fabs from heavy-chain CDR2 insertion libraries

The length of the heavy chain complementarity-determining region two (HCDR2) of the unmutated anti- p-azophenylarsonate (Ars) monoclonal antibody (36-65 mAb) was extended by three residues in order to test whether this insertion can provide additional contacts between the Ab and the antigen. Two lib...

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Bibliographic Details
Published in:Journal of immunological methods 2002, Vol.259 (1), p.43-53
Main Authors: Parhami-Seren, Behnaz, Viswanathan, Malini, Margolies, Michael N
Format: Article
Language:English
Subjects:
Affinity
Animals
Antibody Affinity - genetics
Antibody engineering
Biological and medical sciences
Complementarity Determining Regions - genetics
Complementarity Determining Regions - immunology
complementarity-determining region 2
Diverse techniques
Fundamental and applied biological sciences. Psychology
Fundamental immunology
Hybridomas
Immunoglobulin Fab Fragments - genetics
Immunoglobulin Fab Fragments - immunology
Immunoglobulin Heavy Chains - genetics
Immunoglobulin Heavy Chains - immunology
Mice
Microbiology
Molecular and cellular biology
Molecular immunology
Mutagenesis, Insertional
p-Azobenzenearsonate
p-azophenylarsonate
Peptide Library
Phage-display
Protein Engineering
Saturation mutagenesis
Techniques
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Summary:The length of the heavy chain complementarity-determining region two (HCDR2) of the unmutated anti- p-azophenylarsonate (Ars) monoclonal antibody (36-65 mAb) was extended by three residues in order to test whether this insertion can provide additional contacts between the Ab and the antigen. Two libraries were generated using 36-65 heavy and light chain genes which were cloned as Fab in the phage-display vector pComb3. In the first library, three randomized amino acids were inserted between residues Gly 54 and Asn 55, which are the most solvent exposed residues in the HCDR2 loop. In the second library, in addition to the 3-mer randomized insertion, the flanking residues at positions 54 and 55 were also randomized to allow additional loop flexibility for binding to Ars. Solid-phase and solution phase affinity panning were used to select for clones that bind to Ars. Results indicate that diverse 3-mer HCDR2 insertions can be tolerated, and affinities 10-fold higher than germline encoded 36-65 Ab can be obtained. The sequence diversity of the insertion among the selected clones from both libraries suggests that the insertion increases contact between the Ab and the protein carrier rather than the hapten alone.
ISSN:0022-1759
1872-7905
DOI:10.1016/S0022-1759(01)00488-4