Antigen-antibody binding kinetics for quantitative molecular diagnostics

Abstract

The binding of antibody and antigen constitutes the basis of immunoassays. In this thesis, we aim to explore this binding through three perspectives: a) improving the kinetics of the reaction by increasing mass transport, b) developing a novel method to study the kinetics of binding, and c) using immunoassays for quantifying surface antigen on tumors to uncover tumor heterogeneity. Nowadays, immunoassays are mostly performed on surfaces, which facilitates washing steps and exchanging reagents. A key limitation of such assays is the formation of a layer depleted of analyte, which reduces the reaction rate and thus decreases the overall sensitivity of the test. In the first part of this thesis, we explore the most common current strategies to reduce the depletion layer, i.e., the use of shakers. We show that while shakers are easy to use, they often offer unpredictable results. Such results can be greatly improved through the use of microfluidics, which offers the possibility to control mass transport through convective flows, improving signal while reducing total assay time. Nevertheless, microfluidics has not been adapted to the most common substrates used for immunoassays, microtiter plates. Thus, we propose a new microfluidic concept for controlling the mass transport in microtiter plate wells, which through the use of different kinetic zones allows the exploration of samples with high dynamic ranges in a single test. We then develop a method to evaluate the kinetics of antibody binding using fluorescence lifetime imaging microscopy. Such a method allows a characterization of kinetic constants by comparing the fraction of bound to unbound antibodies on a variety of substrates. We demonstrate its use in breast cancer derived cell blocks and in cancer tissues. Finally, we explore the heterogeneity in cancer, i.e., the different phenotypical profiles that cells in cancer show. Studying tumor heterogeneity is critical to understand cancer evolution in a patient and thus be able to perform accurate patient stratification. We develop a method easily implementable in pathology labs that uses the kinetics of binding of antibodies through immunohistochemical stains to quantify the presence of antigens on different regions of a sample. By performing this localized quantification, we can also explore the heterogeneity present on the tissue. We further investigate heterogeneity using a workflow involving the local tissue lysis and antibody microarray analysis. This method shows a higher capacity of multiplexing, which allowed us to investigate the relative variations of 13 proteins and their inter and intra-tumoral heterogeneity, highlighting the presence of multiple phenotypical variants in a single patient. Thus, in this thesis, we show several use scenarios of antibody-antigen binding kinetics, which highlight the potential of this simple binding to provide us with powerful techniques.