Entwicklung eines Westernblot-Testsystems zur Detektion der humanen Transkriptionsfaktoren GATA-3 und T-Bet
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Philipps-Universität Marburg
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Abstract
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.
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Dates
Created: 2018Issued: 2018-09-24Updated: 2018-09-24
Faculty
Medizin
Publisher
Philipps-Universität Marburg
Language
ger
Data types
DoctoralThesis
Keywords
HumanmedizinT-Betimmune systemwestern blotPhänotypepersonalized medicineTh1 cellTh2-Zellepersonalisierte Medizinbronchial asthmaGATA-3transcription factorImmunsystemTh1-Zellehuman medicineTh2 cellTranskriptionsfaktorBronchialasthmaT-BetphenotyGATA-3Westernblot
DFG-subjects
MedizinBronchialasthmaImmunoblot, GATA-3, T-BetImmunsystemTranskriptionsfaktor
DDC-Numbers
610
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Scheffer, Lukas: Entwicklung eines Westernblot-Testsystems zur Detektion der humanen Transkriptionsfaktoren GATA-3 und T-Bet. : Philipps-Universität Marburg 2018-09-24. DOI: https://doi.org/10.17192/z2018.0342.