This dissertation examines the fundamental principles and applicability of the kinetic exclusion assay (KinExA), developed and marketed by Sapidyne Instruments of Boise, Idaho, since 1995. Chapter One reviews and consolidates the manufacturer’s guidance and many early papers that delineate the practical and theoretical aspects of the technology. In brief, KinExA is a two stage analytical system. In stage one, a number of solutions are prepared, whereby one of the partners is kept constant (the constant binding partner, or CBP), while the other (the titrant) is varied, usually in serial dilution. As the titrant is increased, the free CBP decreases, and is analyzed by a sophisticated and precise microfluidic fluorometric device (stage two). The resulting signal can be related mathematically to the affinity (KD) of the two molecules for each other, and to the kinetic parameters of binding (kon) and dissociation (koff). A comparison of KinExA with other current technology available for quantification of interactions is provided.
In Chapter Two, I investigate the use of KinExA technology with DNA aptamers. DNA aptamers are short nucleotide oligomers selected to bind a target ligand with affinity and specificity rivaling that of antibodies. These remarkable features make them promising alternatives for analytical and therapeutic applications that traditionally use antibodies as biorecognition elements. Numerous traditional and emerging analytical techniques have been proposed and successfully implemented to utilize aptamers for sensing purposes. In this work, I exploited the analytical capabilities offered by the KinExA technology to measure the affinity of fluorescent aptamers for their target molecule thrombin, and quantify the concentration of analyte in solution. Standard binding curves constructed by using equilibrated mixtures of aptamers titrated with thrombin were fitted with a 1:1 binding model and provided an effective KD of the binding in the sub-nanomolar range. However, the experimental results suggest that this simple model does not satisfactorily describe the binding process; therefore, the possibility that the aptamer is composed of a mixture of two or more distinct KD populations is discussed. The same standard curves, together with a four-parameter logistic equation, were used to determine “unknown” concentrations of thrombin in mock samples. The ability to identify and characterize complex binding stoichiometry, together with the determination of target analyte concentrations in the pM–nM range, supports the adoption of this technology for kinetics, equilibrium, and analytical purposes by employing aptamers as biorecognition elements.
In Chapter Three, I explore complex capture agents in the KinExA system. Liposomes made from purified reagents, or from natural cellular membranes, are attached to the beads used in the KinExA process to capture the analyte. Model molecules representing lipophilic dyes, antibodies, and bacterial toxins were successfully captured by the beads for measurement. Residual free ligand captured after pre-equilibration with membrane components, presented as either liposomes or whole cells, could be quantified, and kinetic parameters determined. By this process the “bi-molecular” interaction of the B subunit of cholera toxin for the ganglioside GM1 incorporated into artificial membranes could be quantified, and shown to be dependent upon the presence of the ganglioside in the membrane. The diffusion into artificial membranes of the lipophilic dye DID could be quantified and shown to be dependent upon the amount of lipid available in the equilibration step. In addition, the bulk affinity of a commercial polyclonal antibody for the surface antigens of their target red blood cells could be evaluated. This membrane immobilization process appears to be generally applicable to any membrane system. Thus, it promises to be valuable for the study of signaling molecules for which purified soluble target cellular components may result in misleading information, or for which soluble fragments are unavailable. Likewise, this process should aid in the search for drugs which mimic or antagonize these signaling ligands.
New Applications for the Kinetic Exclusion Assay (KinExA)
