Entwicklung eines Westernblot-Testsystems zur Detektion der humanen Transkriptionsfaktoren GATA-3 und T-Bet

Bronchial asthma is a chronic inflammatory disease. Based on diverse criteria the affected patient population can be divided into different phenotypes and endotypes. Biomarkers represent an important basis for the stratification of the affected patients and increasingly also for decision making with respect to personalization of the therapy. The present work was part of a collaboration project between the Institute of Laboratory Medicine and Pathobiochemistry - Molecular Diagnostics of the Philipps-University Marburg and the DRG Instruments GmbH. The aim of this project was the development of a preferentially monoclonal antibody based Westernblot detection system against the human transcription factors GATA-3 und T-Bet in human blood samples. A detection system for GATA-3 und T-Bet was already established within the working group for the analysis of these factors in total protein fractions from cell lines using polyclonal antibodies. In this work, that system was successfully adapted for the analysis of other sample materials. With these polyclonal antibodies, both the direct detection of recombinant proteins (GATA-3 und T-Bet) and detection of the native transcription factors in human whole blood were achieved. The monoclonal mouse antibodies developed specifically for this project also enabled the detection of GATA-3 und T-Bet for both, recombinant proteins and native proteins in human blood samples using the Westernblot detection system. With polyclonal as well as monoclonal antibody systems, concentration differences could be detected according to varying sample amounts and artificially generated concentration differences in blood samples by spiking with recombinant protein. Nonspecific signal amplification by the second antibody was minimized by switching to an alternative second antibody, reducing the sample volumes used and use of directly HRP-conjugated mouse monoclonal antibodies. Shortcomings were observed for the α-GATA-3 monoclonal antibodies regarding sensitivity and specificity testing. Recombinant protein from another manufacturer could not be detected by these antibodies. Furthermore, cross- reactivity was observed for these antibodies with recombinant T-Bet protein, which could be avoided by higher antibody dilution. In the final discussion section additional options for test optimization with regard to the materials used and sample preparation are discussed. Further, an evaluation of the established test procedure considering current medical research activities in the context of personalization of diagnosis and therapy in patients with bronchial asthma is provided.