Abstract：
: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.
journal_name
J Immunol Methodsjournal_title
Journal of immunological methodsauthors
Parhami-Seren B,Viswanathan M,Margolies MNdoi
10.1016/s0022-1759(01)00488-4keywords:
subject
Has Abstractpub_date
2002-01-01 00:00:00pages
43-53issue
1-2eissn
0022-1759issn
1872-7905pii
S0022175901004884journal_volume
259pub_type
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