PMID: 9661815Jul 14, 1998Paper

Fine tuning of an anti-testosterone antibody binding site by stepwise optimisation of the CDRs

Immunotechnology : an International Journal of Immunological Engineering
Ari HemminkiK Takkinen

Abstract

We have previously reported specificity improvement of an anti-testosterone monoclonal antibody (3-C4F5) by random mutagenesis of the third complementarity determining regions (CDR3s) and by phage display selection. Here we extend the mutagenesis strategy to the other CDRs and select the mutant libraries using two different approaches in order to further fine-tune the binding properties of this recombinant Fab fragment. To improve the affinity the new mutant libraries were selected by using limiting, decreasing concentrations of biotinylated testosterone (TES) in solution and capturing the binders on streptavidin-coated microtiter plate. The specificity was improved by preincubating the mutant libraries in solution with a high concentration of the most problematic cross-reacting steroid, dehydroepiandrosterone sulfate (DHEAS). In two different light chain CDR1 mutant clones isolated from the affinity pannings, the relative TES affinity was increased over 10-fold while the cross-reactivities to related steroids were preserved at the same level as in the parental combined CDR3 mutant clone. New heavy chain CDR1 and light chain CDR2 mutants showing slightly decreased cross-reactivities were isolated from specificity selections. By...Continue Reading

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Citations

Aug 25, 2001·Journal of Chromatography. a·T K NevanenT T Teeri
Aug 1, 2000·Immunology Today·C A Borrebaeck
Feb 13, 2001·Journal of Microbiological Methods·M R BonenS A Hoffman
Nov 26, 1998·Biosensors & Bioelectronics·K Alfthan
Mar 11, 2011·Nature Biotechnology·Andrew R M BradburyJohn McCafferty
Apr 15, 2005·Molecular Immunology·Tania Romo-González, Enrique Vargas-Madrazo
Aug 6, 2004·Protein Expression and Purification·Michael D ScholleBrian K Kay
Jan 29, 2002·Journal of Molecular Biology·C MonnetJ B Charbonnier
Feb 7, 2019·The Biochemical Journal·Deepti Jain, Dinakar M Salunke
Mar 2, 2011·Journal of Molecular Recognition : JMR·Merja H NiemiMatti Höyhtyä
Mar 27, 2007·Biotechnology Advances·Claudia SheedyJ Christopher Hall
Apr 15, 2005·Analytical Chemistry·Timo PulliKristiina Takkinen
Jun 22, 2002·Science·Sang Bok LeeCharles R Martin

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