Entwicklung und Validierung eines neuen Schnellmessverfahrens für Adrenalin im Blutserum

Development and validation of a new rapid measuring method for adrenaline in blood serum

by Uta Geibel
Doctoral thesis
Date of Examination:2017-10-17
Date of issue:2017-10-10
Advisor:Prof. Dr. Viacheslav Nikolaev
Referee:Prof. Dr. Viacheslav Nikolaev
Referee:PD Dr. Marcus Niebert
Referee:Prof. Dr. Rainer Mausberg
Persistent Address: http://dx.doi.org/10.53846/goediss-6517

 

 

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Abstract

English

Two new rapid methods for measuring adrenaline in human blood serum have been established. Both methods use HEK 293 cells, from which two stable cell lines were developed - one cell line was transfected with the Epac1-camps sensor (HEK 293 Epac1-camps) and one with an α1B- adrenergic receptor (HEK 293 α1B). Adrenaline levels are determined indirectly– via binding of adrenaline on a specific adrenergic receptor (β2 and α1B) at the constructed HEK 293 cells and the subsequent release of the second messengers cAMP (HEK 293 Epac1-camps via β2 adrenergic receptor) and respectively Ca2+( HEK 293 α1B via α1B adrenergic receptor). Intracellular cAMP release was detected by measuring changes in FRET-ratio, which occurs when cAMP binds on the cyclic nucleotide binding domain of the Epac1-camps sensor which is coupled with the fluorescent proteins yellow fluorescent protein YFP and the cyan fluorescent protein CFP. CAMP-binding causes a lower energy transfer between YFP and CFP, so called FRET, which can be measured by fluorescence microscopy. However Ca2+ release, after cell stimulation with adrenaline, effects a change of fluorescence intensity in fura-2 labeled HEK 293 α1B cells. It is caused by the formation of a chelate complex consisting of fura-2 and Ca2+, which has a different fluorescence intensity than free fura-2. With the help of these two methods it is possible to indirectly determine adrenaline levels. After testing the transfected cells and preparing dose response curves for each method for reading the sensitivity and evaluating unknown adrenaline samples, both methods were applied to human blood serum. The data shows that it is possible to determine an unknown concentration of adrenaline in human blood serum using both methods. Compared with other methods and procedures the cAMP measurement has an advantage in measuring time. Moreover it is to a small extent superior to the Ca2+ measurement in sensitivity and stability. These findings form an interesting basis for further investigation and standardization in particular concerning the cAMP measurement via FRET. Furthermore the intracellular release of cAMP or Ca2 + in the signal transduction is not only be caused by adrenaline but also by other molecules, so these established methods can be used in the diagnosis of other molecules.
Keywords: CFP; YFP; fura-2; FRET; calcium; cAMP; adrenaline
Schlagwörter: CFP; YFP; fura-2; FRET; Calcium; cAMP; Adrenalin
 
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