Comparison of affinity ranking and immunochemical key data as measure for molecular antibody evolution
The evolutionary optimization of antibody binding propertiesin vitroopens new perspectives for immunochemistry, since the affinity and selectivity of a given antibody molecule can be tailored to meet the requirements of the envisaged analytical application. An efficient strategy for molecular antibody evolution is described that combines randomized point mutations and sequential recombination of variable antibody gene repertoires employing a group-selective library. This strategy enabled significant improvements in the binding of the model analyte atrazine that were monitored by both, kinetic measurements by the optical sensor BIAcore 2000TMand immunochemical key data obtained by enzyme-linked immunosorbent assay (ELISA). The KDof the template antibody IPR-7 was improved by a factor of 17 from 1.27×10–8M to 7.46×10–10M for the optimized variant IPR-83. The enhanced KDis well in line with the 15 fold lowered IC50of the atrazine ELISA, which was shifted from 13.6 µg/l for IPR-7 to 0.9 µg/l for IPR-83. Once the analytical properties of antibody fragments are optimized, antibody functionality can be tailored for specific technical demands, e.g. the directed immobilization on microchip surfaces. As an example, the variable region encoding genes of the scFv variants were subcloned into the Fabfragment expression vector pASK99, in order to reconstitute the antigen binding site of native antibody molecules. The expressed Fabfragments provide a C-terminal affinity tag for functionalized sensor surfaces. Again, the evaluation by ELISA as well as by BIAcore revealed a consistent ratio of analyte binding enhancement for the engineered Fabfragments.