Protein Engineering, Vol 11, 311-319, Copyright © 1998 by Oxford University Press
A Hemminki, S Niemi, AM Hoffren, L Hakalahti, H Soderlund and K Takkinen
The monoclonal antibodies so far developed by hybridoma technology have not
had high enough specificity or affinity to distinguish the closely related
steroid hormones in routine clinical assays. We have employed random
mutagenesis and phage display approaches to improve the specificity of one
anti-testosterone monoclonal antibody (3-C4F5). The affinity of the
antibody is 0.3 x 10(9) M(-1) and the cross- reactivities with most of the
related steroids are low. However, the antibody cross-reacts about 1% with
dehydroepiandrosterone sulfate (DHEAS) and owing to the high DHEAS serum
concentration this is about 1000-fold too high for clinical immunoassays.
The complementarity- determining regions (CDRs) of the heavy and light
chains, which were predicted by molecular modelling to be in close contact
with the testosterone (TES) ligand, were randomized and mutant Fab
libraries were cloned into a phagemid vector. Binders were selected by a
competitive panning procedure. By combining the identified light and heavy
chain CDRIII mutations the TES affinity was preserved at the wild- type
level but DHEAS cross-reactivity was decreased to 0.03%. An important
finding was that by the competitive panning procedure the overall binding
specificity of the 3-C4F5 antibody was refined, since the
cross-reactivities to related steroids were also significantly decreased in
the combined mutant.
ARTICLES
Specificity improvement of a recombinant anti-testosterone Fab fragment by CDRIII mutagenesis and phage display selection
VTT Biotechnology and Food Research, Espoo, Finland.
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