Evolutionary Affinity and Selectivity Optimization of a Pesticide-Selective Antibody Utilizing a Hapten-Selective Immunoglobulin Repertoire
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- 作者：Karl Kramer
- 刊名：Environmental Science & Technology
- 出版年：2002
- 出版时间：November 15, 2002
- 年：2002
- 卷：36
- 期：22
- 页码：4892 - 4898
- 全文大小：139K
- 年卷期：v.36,no.22(November 15, 2002)
- ISSN：1520-5851
文摘
Selectivity and sensitivity are considered as pivotalcriteria for the quality of immunochemical assay designsin environmental analysis. They are essentially determinedby the variable domains of the implemented antibody.The variable domains of a triazine-selective single-chainFv (scFv) were genetically engineered by stringent molecularevolution in order to optimize analytical characteristicsof the corresponding atrazine immunoassay. Gene variationof the template antibody by sequential shuffling againstthe variable heavy and light chain repertoire of a triazine-selective immunoglobulin library was enhanced byintroducing additional point mutations. Improved scFvvariants were selected by phage display employing anatrazine derivative. By this means the paramounting affinityof the initial scFv to sebuthylazine was shifted for themutant antibodies toward a preferential recognition of theenvisaged target analyte atrazine. In addition, the detectionlimit of the atrazine assay was significantly improved by factor25 from 5.1 
g/L for the initial template antibody to 0.2g/L for the mutant antibodies. The contribution of theengineered antibody variants to the assay improvement isalso reflected by a shift of the equilibrium dissociationconstant KD from 1.27 × 10-8 M of the template antibodyto 7.46 × 10-10 M of the optimized variant. Sequenceanalysis revealed a bias of amino acid substitutions in thefirst two complementarity-determining regions (CDR) andthe flanking framework regions of both variable chains forthe shuffled clones as well as a deletion in the CDR3 ofthe light chain. Particularly the mutations of the VL domainturned out to have a decisive impact on the alterationsin the analytical performance of the engineered scFv mutants.The application of the mutant antibodies for the atrazinedetermination of soil samples revealed consistency with HPLCdata within the experimental error.
g/L for the initial template antibody to 0.2g/L for the mutant antibodies. The contribution of theengineered antibody variants to the assay improvement isalso reflected by a shift of the equilibrium dissociationconstant KD from 1.27 × 10-8 M of the template antibodyto 7.46 × 10-10 M of the optimized variant. Sequenceanalysis revealed a bias of amino acid substitutions in thefirst two complementarity-determining regions (CDR) andthe flanking framework regions of both variable chains forthe shuffled clones as well as a deletion in the CDR3 ofthe light chain. Particularly the mutations of the VL domainturned out to have a decisive impact on the alterationsin the analytical performance of the engineered scFv mutants.The application of the mutant antibodies for the atrazinedetermination of soil samples revealed consistency with HPLCdata within the experimental error.