Immobilisierung von Derivaten des Influenza-A-Neuraminidase-Inhibitors GS4071 zur Anreicherung von Influenza-A-Virus-Neuraminidase an Polymeroberflächen
Neuraminidase inhibitors represent an important group of chemotherapeutics for the treatment of influenza diseases. Since the treatment is most effective during an early stage of the disease, fast and reliable means of diagnostic are needed. Most of the currently available test kits are based on the recognition and binding of antibodies to certain surface proteins of the virus. As such the kits are subject to structural changes in those proteins by antigenic drift. The aim of this work is to lay the foundation for a non-immunological diagnostic device. This is achieved by preparing derivatives of a neuraminidase inhibitor (GS4071) that can be coupled to a polymer surface either covalently or by adsorption. Based on a precursor of the inhibitor two derivatives with a different (-unsaturated alkylspacer (C-9 and C13) and a biotinylated derivative (BID) were synthesized and characterized. These derivatives were immobilized either covalently on glass plates or silicone foils by Hydrosilylation or to a polystyrol surface by specific Biotin-Avidin interaction. Covalent coupling is conducted by reacting the (-double bond of the alkyl derivatives with the silane functionalized surfaces of glass and silicone. Successful covalent coupling and the degree of occupancy of glass and silicone with the two derivatives are confirmed by X-ray photoelectron-spectroscopy (XPS) and contact angle measurements. Polystyrene surfaces for adsorptive immobilization are cleaned, treated with streptavidin, blocked with BSA and incubated with BID. The prepared surfaces were then being tested for their ability to bind recombinant Influenza Virus Neuraminidase (NA). The assays were conducted following the ELISA (enzyme linked immunosorbent assay) principle. The surfaces with the immobilized derivatives were incubated with the recombinant NA and the amount of bonded NA was measured. Despite the fact that both derivatives used for covalent coupling were able to bind to NA in solution, after coupling no binding neither on glass nor on silicone was detected. This is possibly due to the hydrophobic nature of the spacer and its tendency to interact with the hydrophobic parts of the surfaces. Adsorptive immobilization of BID by the avidin-biotin system and subsequent binding of NA proved to be successful. This system has a detection limit of 3 ng NA/ml and thus proves the descried principle to be feasible.
info:eu-repo/classification/ddc/540, Chemie, Influenza-A-Virus, Neuraminidase, Enzyminhibitor, Immobilisierung, Polymere
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