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1	Towards Development of an Immunoassay Utilizing Circularly Permutated Proteins to Detect Environmental Contaminants Zunnoon Khan, Sara 29 August 2013 (has links) A fusion protein composed of antibody fragments and β-lactamase was earlier created by Kojima et al. (2011), with antigen specificities against a bone disease marker and a pesticide. The enzyme was circularly permutated and fused to the variable heavy and light chain antibody fragments, thereby ensuring inactivity until binding of the target antigen triggered enzyme activation. Upon activation, the β-lactamase produced a colorimetric signal, which indicated antigen presence. In this work, a similar strategy was used to create two novel fusion proteins composed of circularly permuted β-lactamase and superfolder green fluorescent protein with anti-benzo[a]pyrene variable antibody fragments. The fusion proteins were designed and expressed in E. coli for the development of a single-step visual immunoassay. It was hypothesized that the cp reporter proteins would be activated once the binding of B[a]P to the variable antibody fragments occurred, and this interaction was expected to produce a detectable colorimetric or fluorescent signal. Although positive results were obtained in one instance, substantial supportive evidence in favour of the hypothesis could not be obtained. / SENTINEL Bioactive Paper Network, Natural Sciences and Engineering Research Council of Canada (NSERC), Canada Research Chairs Program. immunoassay B[a]P benzo[a]pyrene circular permutation circularly permutated environmental contaminant beta-lactamase superfolder GFP green fluorescent protein fluorescence nitrocefin cross-reactivity imidazole carcinogen detection method fusion protein colorimetric reaction scFv anti-B[a]P scFv cp

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Towards Development of an Immunoassay Utilizing Circularly Permutated Proteins to Detect Environmental Contaminants