NR ATVH
AU Boesen,M.
TI Entwicklung und Anwendung eines enzymimmunologischen Verfahrens zum Nachweis von zellulärem Prion Protein bei Wiederkäuern
QU Inaugural-Dissertation zur Erlangung der tiermedizinischen Doktorwürde der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München von Melanie Boesen aus München, USA, München, 15. Juli 2005
IA http://edoc.ub.uni-muenchen.de/archive/00004014/
PT Dissertation
AB
This paper deals with the development of an enzyme-immunosorbent-assay (EIA) for the detection of cellular PrP (PrPc) as well as its application for the analysis of diverse tissues derived from cattle, calves and lambs. Additionally, milk from cows, goats and sheep was analysed by using this test system for the occurrence of PrPc and its distribution in milk fractions.
With respect to the establishment of a sandwich-EIA, the suitability of several monoclonal antibodies was tested. Aiming at the development of a rapid and simple extraction procedure for the detection of PrPc in tissues, brainstem homogenates of cattle and sheep were produced by using different extraction buffers and subsequently analysed by the optimized EIA-system.
After optimization of further parameters, the following standard procedure was used for the examination of the tissue samples. By using a RiboLyser, tissues were homogenized in PBS containing 3 % (w/v) glucose and subsequently incubated at 37 ° for 40 min to improve the detectability of PrPc. Proteinase K digested samples served as negative controls.
During analysis of the tissue samples it turned out that prion proteins are detectable in nearly every tissue. Besides brainstem other organs and tissues, respectively, are rich in PrPc. Particularly, the third eyelid of lambs, lungs of calves and lambs as well as the cortex of kidneys of adult and juvenile cattle contained high amounts of PrPc. Comparatively low concentrations of prion protein could be detected in muscles and lymphatic organs of the animals. For some organs, such as the liver, high variability of the PrPc content was found between the examined individuals.
Detectable amounts of PrPc were also found in milk and its fractions (skimmed milk, whey and casein-fraction), which could be applied to the EIA without further treatment. PrPc could be detected in milk from sheep, goats and cows. The analysis of fractionated ewes? milk showed that defatting of the samples had no influence on the detectable PrPc-content and that the predominant PrPc-portion is found in the whey-fraction.
AD Melanie Boesen aus dem Institut für Hygiene und Technologie der Lebensmittel tierischen Ursprungs (Lehrstuhl: Prof. Dr. E. Märtlbauer) der Tierärztlichen Fakultät der Universität München
SP deutsch
PO Deutschland