| The work described in this dissertation was done as a part of a project funded by the American Water Works Association Research Foundation (AWWARF). The goal of this project was to create a portable device that could detect all types of biological warfare agents (BWA)---bacteria, spores, viruses and toxins---in finished water.; Here, I develop an immunoassay for detecting B. anthracis spores simulant, B. globigii (BG) spores, test individual water components as potential assay interferents, evaluate different detection possibilities, investigate the use of commercial PAMAM dendrimers to improve the assay sensitivity and develop a simulant alternative to BG spores---BG Bugbeads.; A bead-based enzyme labeled sandwich immunoassay for BG spores was developed and optimized. The total assay time was 45 min, and the detection limit was 2.6 x 103 spores/mL or 7 spores in the final detection volume. No major assay interference from eight individual components of finished water was seen. BG spores were successfully detected in Cincinnati finished water.; Two immunoassay detection modes were evaluated: fluorescence detection, and electrochemical detection by the microelectrode. The alkaline phosphatase (AP)-fluorescein diphosphate couple was chosen for the immunoassay from eight enzyme-substrate couples tested for its superior kinetic parameters and low detection limits. Immunoassays for three antigens---mouse immunoglobulin (IgG), ovalbumin and mouse IgG Bugbeads had detection limits similar or superior to those obtained with electrochemical detection.; Commercial PAMAM dendrimers were used as linkers between the capture beads and the primary antibodies to enhance the sensitivity of the immunoassay. The immunoassay with G5.5 dendrimers as linkers had a higher sensitivity and lower detection limits than the assay without a linker. The dendrimers enhance antigen capture by increasing the beads capacity for the primary antibody and by providing more conformational freedom to the latter.; BG Bugbead---an artificial spore---was developed as an alternative simulant to BG spores. Two methods of spore protein extraction were compared: extraction by mechanical grinding and by chemical decoating. The immunoassay for BG Bugbeads had a detection limit comparable to that obtained in the assay of the same format for BG spores. |
