Development of New and Modification of Existing Methods for the Primary Screening of Aptamers and Antibodies by Means of Capillary Electrophoresis

Discovery of new drugs starts with screening a library of drug candidates (ligands) for their ability to bind targets. Such a screening can be conducted by a variety of analytical techniques; however, Capillary Electrophoresis (CE) is the the most preferably one. CE provides homogenous separation of the species, generates minor amount of waste and can serve both analytical and preparative purposes. Sometimes, CE-based screening approaches fail due to insufficient separation of target-ligand complexes (TL) from non-binding ligands or due to the inability to collect the complexes. The first problem affects all types of ligands, but the second one is especially acute for the oligonucleotides, since oligonucleotide binders are often required to be collected for the further amplification and sequencing. To address this problem, I developed a mathematical model that allows precise prediction of elution times of protein-oligonucleotide complexes, which guarantees their successful collection. Such a predictor is especially useful for the cases when the complexes are present in the undetectably small amount. The developed model significantly advances the existing screening approaches, such as NECEEM (Non-equilibrium Capillary Electrophoresis of Equilibrium Mixtures) and Ideal Filter CE (IFCE). The problem of inefficient separation of TL complexes from the initial targets and ligands is especially acute for the field of CE-based immunoassays. Particularly, for the assays that study virus-antibody interactions. The existing assays provide a minor (almost negligible) separation of virus-antibody complexes from the source molecules and allow screening mixtures of antibodies and spherical viruses only. In order to address these problems, I developed an assay for screening bivalent antibodies for their ability to bind another common type of viruses, which are rod-like viruses. The developed assay was able to provide a baseline separation of virus-antibody complexes from free viruses and antibodies. In addition to that, my work resulted in the discovery of the previously unknown phenomenon of abnormally high electrophoretic mobility of the complexes between rod-like virions and bivalent antibodies. I explained this phenomenon and built a simple mathematical model for the prediction of the number of cross-linked virions in the complexes.