Fine tuning of an anti-testosterone antibody binding site by stepwise optimisation of the CDRs
被引:33
作者:
Hemminki, A
论文数: 0引用数: 0
h-index: 0
机构:VTT Biotechnol & Food Res, FIN-02044 Espoo, Finland
Hemminki, A
Niemi, S
论文数: 0引用数: 0
h-index: 0
机构:VTT Biotechnol & Food Res, FIN-02044 Espoo, Finland
Niemi, S
Hautoniemi, L
论文数: 0引用数: 0
h-index: 0
机构:VTT Biotechnol & Food Res, FIN-02044 Espoo, Finland
Hautoniemi, L
Soderlund, H
论文数: 0引用数: 0
h-index: 0
机构:VTT Biotechnol & Food Res, FIN-02044 Espoo, Finland
Soderlund, H
Takkinen, K
论文数: 0引用数: 0
h-index: 0
机构:VTT Biotechnol & Food Res, FIN-02044 Espoo, Finland
Takkinen, K
机构:
[1] VTT Biotechnol & Food Res, FIN-02044 Espoo, Finland
[2] Or Diagnost, FIN-90460 Oulunsalo, Finland
[3] Orion Diagnost, FIN-20101 Turku, Finland
来源:
IMMUNOTECHNOLOGY
|
1998年
/
4卷
/
01期
关键词:
CDR mutagenesis;
competitive and affinity panning;
recombinant Fab;
specificity and affinity improvement;
testosterone;
D O I:
10.1016/S1380-2933(98)00002-5
中图分类号:
Q81 [生物工程学(生物技术)];
Q93 [微生物学];
学科分类号:
071005 ;
0836 ;
090102 ;
100705 ;
摘要:
Background: 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. Objectives: 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. Study design: 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). Results: 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 combining compatible mutant CDRs together we were able to create a Fab fragment with over 12-fold higher relative TES affinity and significantly lower cross-reactivity to DHEAS when compared to the original monoclonal antibody 3-C4F5. Conclusions: Our results demonstrate that a high-affinity and selective recombinant Fab fragment working over a wide TES concentration range with clinical samples could be generated by CDR mutagenesis and phage display selection. (C) 1998 Elsevier Science B.V. All rights reserved.