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Studies of acridinium ester labeled antibodies for chemiluminescence detection in affinity chromatography
Abstract
The work in this dissertation investigates antibodies labeled with acridinium ester for use in post-column detection for high-performance immunoaffinity chromatography (HPIAC). The areas that were studied included the optimization of reaction conditions for labeled antibodies and the use of various surfactants to increase the luminescence signal for labeled antibodies and free phenyl acridinium ester. Also, chromatographic equations that predict the behavior of simultaneous addition chromatographic immunoassays were developed and were examined. Chapter one explains various background information related to the topics studied, such as antibody function and structure, acridinium esters, chemiluminescence, and affinity chromatography. Chapter two deals with improving chemiluminescence detection by optimizing the reaction conditions for acridinium ester labeled antibodies. In this study, various factors that influence light production were examined, such as hydrogen peroxide, sodium hydroxide, and surfactant concentrations. The luminescence was measured by using either a benchtop luminometer or a post-column reactor, and the results were compared between these two methods of detection. Chapter three explores the use of various surfactants for increasing the luminescence intensity of labeled antibodies and phenyl acridinium ester samples at pH 2.5 and 7.4. In this study seven surfactants were investigated, including cationic, anionic, zwitterionic, and nonionic compounds that are commonly used in the laboratory and are readily available. All luminescence measurements were done using a benchtop luminometer using hydrogen peroxide and sodium hydroxide as the initiating reagents. It was determined that five of the surfactants increased the luminescence intensity, while two compounds decreased the signal for both samples. Chapter four explains a theoretical model for determining the behavior of Simultaneous addition chromatographic immunoassays. In this study, equations were developed that predict calibration curves for competitive binding chromatographic immunoassays when certain factors are varied, such as the flow rate, amount of label, and number of binding sites in the column. In addition, various aspects of each assay were compared, including the limit of detection and dynamic range.
Subject Area
Analytical chemistry
Recommended Citation
Reiter, Wanda Sue, "Studies of acridinium ester labeled antibodies for chemiluminescence detection in affinity chromatography" (1997). ETD collection for University of Nebraska-Lincoln. AAI9730280.
https://digitalcommons.unl.edu/dissertations/AAI9730280