Combinatorial search for diagnostic agents and application of affinity capillary electrophoresis in high-throughput library screening.

摘要

Combinatorial chemistry approach was employed in the search of tight-binding ligands for the development of a novel Lyme-disease diagnosis assay using affinity capillary electrophoresis. Lyme-related monoclonal anti-flagellin antibodies H9724 and anti-OspB antibody 84c were chosen as two systems. By using the combination of split synthesis and chain termination strategy, encoded libraries containing 1.4 millions of peptides in each library were constructed. A high-throughput on-bead ELISA was employed to screen these libraries against the two antibodies respectively. By decoding the ligand candidates by MALDI-MS, tight-binding ligands were rapidly identified. A capture ELISA was used to evaluate the new ligand in solution. Some of the newly identified ligands showed at least 10 times improvement in binding to antibody 84c. Through the strategy of deconvolution, four best-binding ligands for H9724 were obtained from a biased ligand library. Therefore, combinatorial chemistry was proven as an effective approach for the rapid identification of tight-binding ligands.;In this dissertation, the applications of combinatorial chemistry in ligand optimization and cross-reactive study were also explored using these two Lyme disease related antibodies. It indicated that the template of library played a key role in ligand optimization. However, combinatorial chemistry demonstrated its powerful potential in the search of cross-reactive epitopes as our in vitro experiments indicated 84c could cross react to a human protein PLC-beta2.;In second part of the dissertation, a multiple-injection capillary electrophoresis (CE) technique is described. It was employed in the development of enzyme kinetic assay using dipeptidase VanX as an example. VanX is involved in bacterial vancomycin resistance and an intensive research is undergoing to combat the resistant bacteria. A multiple-injection CE-based VanX assay was developed and validated using four substrates. Kinetic parameters obtained by this new method were comparable with the data obtained by the conventional assay.;Combining the power of affinity capillary electrophoresis with multiple-injection technique, a novel method with potential for high-throughput library screening was developed using vancomycin-peptide interaction as a model system. The advantages and limitations of this method are discussed in this dissertation to shed a light in further development of this potentially powerful technique.