Immunoassays represent an important analytical tool in diagnostics and research. Since the production of antibodies is often time-consuming and costly, there is a need for the development of alternative detection systems. The aim of this master's thesis was to develop a modular detection probe based on a recombinant filamentous bacteriophage as an alternative to antibodies in enzyme-linked immunosorbent assays. The hybrid bacteriophage vector f88KE, which enables dual display, was used to simultaneously display the peptides SpyTag and SnoopTag on the surface of the filamentous bacteriophage. The reporter component of the system consisted of the fusion protein SpyCatcher-betalactamase, composed of the protein partner SpyCatcher and the reporter enzyme betalactamase. The binding component was represented by various binding protein-SnoopCatcher fusion proteins composed of different antibody mimetics and the protein partner SnoopCatcher. The fusion proteins were expressed in Escherichia coli, isolated, and purified using chromatographic methods. The successful production of the fusion proteins and their conjugation to recombinant filamentous bacteriophages were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting. The functionality of the developed system was assessed by detecting a model antigen and inflammatory cytokines using a modified phage enzyme-linked immunosorbent assay and a sandwich enzyme-linked immunosorbent assay format. All designed fusion proteins were successfully expressed, isolated, and purified and were subsequently conjugated to the surface of recombinant filamentous bacteriophages using two orthogonal Catcher/Tag systems. The doubly conjugated bacteriophages specifically bound their targets and generated a measurable signal. However, comparison with established immunochemical assays revealed that the sensitivity of the developed system was approximately three orders of magnitude lower than that of a conventional enzyme-linked immunosorbent assay. Nevertheless, the developed detection probe represents a promising modular platform for the detection of various biological molecules. The results demonstrate the potential of recombinant filamentous bacteriophages as functional alternatives to antibodies, while further optimization of the reporter system and detection conditions could improve sensitivity and expand the applicability of the developed platform in analytical, research, and diagnostic applications.
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