Preparation of enzyme - analyte conjugates containing linker arms

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Title: Preparation of enzyme - analyte conjugates containing linker arms
Author: Zhu, Tong
Abstract: In this study, we are examining the effect of the length and polarity of peptide linker arms on the performance of enzyme - analyte conjugates in a reagentlimited heterogeneous immunoassay which utilizes a solid phase format. In our model system, our goal is to attach benzoylecgonine, the primary urinary metabolite of cocaine, to glucose oxidase by a series of peptide linker arms. Peptide linker arms were synthesized using the fluorenylmethoxycarbonyl (Fmoc) solid phase peptide synthesis strategy described by Stewart and Young (1). We attached the BEC to the exposed amino - terminal end of the peptide linker arm by the free carboxyl group of BEC prior to removal of the peptide from the solid phase to avoid the requirement for isolation of the peptide before reacting with the analyte. The BEC-peptide will be activated by the mixed anhydride method (2) and coupled to glucose oxidase. The BEC - peptide can also be conjugated to the carbohydrate portion of glucose oxidase using peptides containing carboxy - terminal hydrazides. The enzyme-analyte conjugates will be characterized immunochemically to determine the effect of the linker arm on conjugate performance in an immunoassay. We will focus mainly on the following three factors: binding of the conjugate to the antibody in the absence of benzoylecgonine; displacement of the bound enzyme - analyte conjugate by the physiological concentrations of the analyte; and non - specific binding. It is expected that the enzyme analyte conjugate containing the linker arm system should give better binding of the antibody to the enzyme - analyte than the one without any linker arm. This system for binding an analyte to an enzyme through a peptide linker arm could be expanded to a variety of applications in the future.
Record URI: http://hdl.handle.net/1850/12161
Date: 1995-05

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