Diagnostische Fusions- und Linkerproteine zur Entwicklung neuartiger Immunoassays

In dieser Arbeit wurden Linker-/Fusionsproteine rekombinant hergestellt und getestet. Sie dienten der Entwicklung neuartiger Analyseverfahren, sowie der Verbesserung bereits bestehender antikörperbasierter Analyseverfahren. Hierbei wurde vor allem die Eigenschaft des Concanavalin A Proteins genutzt,...

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Bibliographic Details
Main Author: Dassinger, Nina
Contributors: Keusgen, Michael (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2015
Online Access:PDF Full Text
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n this work different linker / fusion proteins were produced recombinantly and tested . These served the development of new analysis methods , as well as the improvement of existing antibody-based methods of analysis . In this case, especially the property of the protein Concanavalin A was used to immobilize proteins on surfaces which have been previously coated with a sugar compound . The second domain of the proteins was adjusted depending on the use .The universal, bifunctional linker protein ConA SAv should serve the development of an enrichment module for purification of analytes from complex matrices. For this purpose the gene was the concanavalin A binding domain C-terminal fused by alanine linker to the gene of streptavidin binding domain, to allow the connection of biotinylated antibody. The expression was performed in E. coli and assayed the functionality of the protein using several methods. The result of the co-immunoprecipitation demonstrated the full functionality of the linker protein ConA SAv; both functional domains are binding-active. The recoverability of the dextran matrix in the form of Sephadex® G-25 beads could also be detected. The results of the measurements are to be regarded RIfS also positive. The linker protein alone could easily of a functionalized dextran chip surface, to be replaced by a competing sugar. However, it was the regeneration of the linker protein, wherein binding of the biotinylated antibody limited. To further investigate SPR measurements were conducted. This confirmed the observations that have been made at the RIfS. The linker protein was no longer to be solved in connection of a biotinylated antibody from the surface. One approach was to generate various mutants. For this purpose, the binding pocket of ConA was modified at the genetic way. The mutants D90N and R110L were also tested, and it was found that the binding activity was either questionable or no longer exists.In the further course fusion proteins were developed for the disease diagnosis. For this special surface antigens were selected, against which the immune system, especially in the early phase of infection, while the IgM response, antibody forms. After literature review, the surface protein C (OspC) and an epitope (C6) of VlsE antigen as a good choice turned out. Both cDNA sequences of the epitopes were respectively C-terminal, coupled through a short linker sequence to the cDNA sequence of the ConA and subsequently produced in E. coli recombinant. After expression, the fusion proteins by SPR and RIfS were tested for their functionality. The results of the SPR measurements showed the functionality of both fusion proteins. The fusion proteins bound to the functionalized gold surfaces with mannan and specifically detect the antibodies from the test sera. The measurements by RIfS could only confirm these observations. However, particularly the ConA-OspC protein proved to be very stable. Therefore, in regard to the use in commercially available products, the storage stability, evaluated in lyophilization of the two proteins. These studies were performed by ELISA measurement series. In the ELISA measurements both proteins showed a very high significance in the detection of Lyme disease-antibodies from the blood serum. They also retained after lyophilisation their ability to function, however, confirmed also here again the results of the SPR measurements. ConA-OspC was unlike ConA-C6 considerably less stable and lost heavily on signal strength. However, the signal strength and the storage stability could be improved by the use of special stabilizing reagents.Recently a second universal linker protein has been developed, which made it possible to bind a variety of unlabeled IgG antibody with sugar on functionalized surfaces. For this purpose, the cDNA of a binding domain of protein A C-terminal, coupled through a short linker sequence to the cDNA sequence of the ConA and subsequently produced in E. coli recombinant. The function testing was carried out by SPR, first using a model assay, which was then successfully transferred to a diagnosis in the veterinary field. The detection of acute pancreatitis and pancreatic insufficiency in cats was performed by measuring the fTLI level in blood serum. Using SPR succeeded in creating a calibration of samples of known concentration fTLI, for further investigation of unknown fTLI concentrations in real samples. To illustrate a point-of-care diagnostics, 3D polymer sintered body were functionalized with a sugar compound and the method of analysis has been transferred from the 2D to the 3D surface. Again, succeeded in creating a calibration using known sample concentration. On this basis, a further concentration determination could successfully be carried out in real samples of unknown concentration fTLI.