Antibodies and Antibody Mimics in Binding Assays

Abstract: This thesis presents development of new analytical methods using antibodies and molecularly imprinted polymers (MIPs) in binding assays: 1) The high specificity of antibodies and the wide variety of efficient labels make immunoassay one of the most popular analytical techniques today. Enzyme-linked immunosorbent assay (ELISA) is most widely used, since the enzyme label can be used as a catalyst to provide very high signal amplification. However, non-specific binding of proteins on surfaces may cause erroneous results. This thesis exploits the use of materials such as dextrans and dendrimers to modify surfaces, which are commonly used in ELISA, to reduce the interference of non-specific binding. This was realized by appropriate covalent attachment of the modifiers to the solid supports resulting in remarkable improvements in assay sensitivity and selectivity. 2) Due to the high physical and chemical stabilities, MIPs are increasingly used as alternatives to antibodies as selective binding materials. In general, MIPs are prepared by template-directed polymerization of functional monomers and cross-linkers. During the polymerization, the template guides the assembly of functional monomers in the resulting cross-linked polymer. Upon removal of the template, the polymer retains specific binding sites that can selectively rebind the original template, as well as structurally related molecules such as analyte-enzyme conjugates. In this thesis microspheric MIPs prepared by a precipitation polymerisation have been employed. The molecularly imprinted microspheres displayed favourable binding characteristics and were successfully used in aqueous environments. Novel applications of the MIPs in ELISA and scintillation proximity assay (SPA) were demonstrated. Assays for small molecules such as 2,4-dichlorophenoxyacetic acid and (S)-propranolol using microspheric MIPs proved highly selective and sensitive.