AdvisorWilson, George S.
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PublisherThe University of Arizona.
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AbstractThe optimization of the competitive binding and sandwich immunoassays was investigated in order to examine some commonly encountered experimental aberrations. Experimental and theoretical results were integrated to provide a better understanding of the causes of the "hook" or "prozone" effect. The "hook" generates ambiguous results for the test sample in an immunoassay. The competitive binding assay manifests a low dose "hook" effect while the sandwich immunoassay demonstrates a high dose "hook" effect. Human growth hormone (hGH) having no repeating epitopes was examined as a model for the "hook" effect in the competitive binding assay. Three model analytes, hGH, the dimeric form of hGH (D-hGH, having a discrete number of repeating epitopes) and ferritin (multiple epitopes) with differing immunological properties were employed. To elucidate the low dose "hook" effects in the competitive binding assay the interaction of two different monoclonal antibodies with hGH was investigated. The individual monoclonal antibodies show normal behavior in a competitive binding assay, but mixtures of antibodies demonstrate a "hook" attributed to formation of multicomponent complexes in solution. Size exclusion chromatography was employed identify higher molecular weight complexes. The experimental data were supported by theoretical models assuming a circular tetrameric complex formation. The one-step sandwich immunoassay suffers from the "hook" effect irrespective of the analyte characteristics. Model analytes, hGH, D-hGH, and ferritin offer new insights into general guidelines for assay procedures allowing the analyst to quickly optimize assay conditions without a priori knowledge of the immunological characteristics of the antibody or the antigen. Experimental and theoretical data show that the high capacity solid-phase antibodies shifted the "hook" to relatively higher analyte concentrations. The effect of the concentration of labeled antibody on assay response was examined theoretically. The cause of the "hook" effect in two-step sandwich immunoassay is attributed to the desorption of the bound analyte most likely due to a conformational change after the labeled antibody interacts with the several epitopes of the analyte, hence the assay for hGH shows no "hook" effect. Two different protocols for D-hGH were implemented. These assays demonstrated a "hook" effect if the labeled antibody was allowed to interact with more than one epitope of the analyte. Multiple epitope interactions with the labeled antibody, as exemplified by ferritin, demonstrate the "hook" effect. The effect of the ferritin concentration, capture antibody and the labeled antibody was examined.