Design of Antibody-Based Diagnostics and Affinity Chromatography Systems Utilizing the Nucleotide Binding Site (NBS)
This dissertation focuses on developing advanced diagnostic systems, efficient antibody purification techniques, and novel antibody-drug conjugation methods while aiming to improve availability of antibody-based technologies to end-users. Antibodies have become widely used agents for clinical treatment of cancers and autoimmune diseases due to their high level of specificity and selectivity toward known target molecules (antigens) found to be overexpressed on cancerous cells. Despite the many techniques that utilize antibodies necessitate that they are modified in some way by conjugating functional ligands to them – endowing them with specific functionalities unique for each intended application – they are still in need of improvement. This dissertation addresses the particular technical challenges related with site-specific labeling of antibodies in order to enhance diagnostic and therapeutic platforms. Specifically, there are two main directions of this dissertation – (i) oriented immobilization of antibodies and their Fab fragments to small scaled diagnostic surfaces, such as microfluidic devices and nanoparticles, while preserving their antigen binding activity and structure stability, and (ii) designing a novel affinity chromatography technique for purification of antibodies from complex protein environment that is an easy, efficient and cost effective way. An underutilized, highly conserved binding domain located on the antibody variable fragment, known as the nucleotide binding site (NBS) was implemented for site-specific functionalization of antibodies and antibody Fab fragments, as well as developing antibody purification systems. Through in silico screening of small molecules, ring structured aromatic molecules such as indole-3-butyric acid (IBA), tryptamine, fluorine-4-carboxylic acid (FCA) were identified as moderate binding nucleotide analog with Kd = 1–10 µM to selectively target the NBS. A unique small molecule antibody purification was developed by targeting the antibody NBS with surface immobilized NBS analog, and a stable covalent bond between the antibody NBS and the NBS analog was formed through a photo-chemical reaction utilizing UV energy to homogenous site-specific conjugation of target molecules to the light chains of antibodies and antibody Fab fragments. This NBS-based purification method will potentially contribute to an increasing quality of antibody production as well as lowering the overall cost that is associated with it. Site-specific functionalization and oriented immobilization of antibodies and their Fab fragments for developing enhanced diagnostic applications resulted in designing inexpensive, sensitive, and easy to use detection systems that can be used in underdeveloped countries.
| Author ||Nur Mustafaoglu|
| Contributor ||Basar Bilgicer, Research Director|
| Degree Level ||Doctoral Dissertation|
| Degree Discipline ||Bioengineering|
| Degree Name ||Doctor of Philosophy|
| Submission Date ||2017-09-20|