20.500.12556/RUL-120377 Določanje humanega prionskega proteina PrP226* v krvi Detection of human prion protein PrP226* in blood Celični prionski protein (PrPC) je glikoprotein. V telesu ima več bioloških funkcij, med katerimi so celična adhezija, medcelična signalizacija, vzdrževanje ionske homeostaze in zaščita nevronov. Povezujemo ga tudi z nastankom nevrodegenerativnih bolezni. Po cepitvi PrPC s proteazo ADAM 10 nastane odcepljena oblika PrP brez GPI-sidra. Ta prosto plava po telesnih tekočinah in se veže na PrPSc agregate ter preprečuje njihovo pomnoževanje. Povod za razvoj nevrodegenerativnih bolezni pa je lahko situacija, ko PrPSc agregati vežejo PrPC in se ta pretvori v PrPSc, ki ga potem ADAM 10 sprosti iz membrane. Zaradi te lastnosti se osredotočamo na določanje skrajšane oblike PrP v krvi, s poudarkom na PrP226*, ki je izjemen biomarker za diagnozo prionskih bolezni. Namen dela je bil izdelati zanesljiv presejalni test ELISA za določanje prisotnosti skrajšane oblike PrP (PrP226*) v vzorcih plazme krvodajalcev z uporabo mAb V5B2 kot lovilnega protitelesa ter uporabo mAb E12/2 kot detekcijskega protitelesa. Določili smo občutljivost testa, nato smo testirali 484 vzorcev plazme krvodajalcev. Po presejalnem testiranju smo ugotovili, da zaradi prisotnosti heterofilnih protiteles (HA) dobivamo lažno pozitivne rezultate. Prisotnost HA v vzorcu smo potrdili z uporabo komercialno dostopnih protiteles. Z različnimi metodami smo nato iz vzorca poskušali izločiti HA. Cellular prion protein (PrPC) is a glycoprotein. It has several biological functions in the body, among which are cell adhesion, intracellular signaling, maintenance of ion homeostasis, and protection of neurons. It is also associated with the development of neurodegenerative diseases. PrPC is shed by the ADAM 10 protease, which result in the formation of shed PrP that lack GPI-anchor. It floats freely in body fluids and binds to PrPSc aggregates, preventing them from multiplying. The problem, however, arises when PrPSc aggregates bind PrPC and convert them into PrPSc, which is then released from the membrane by ADAM 10 protease. This can lead to the development of neurodegenerative diseases. Because of this role, we focus on detecting shed PrP in the blood, with focus on PrP226*, which is an outstanding biomarker for the diagnosis of prion diseases. The aim of this work was to develop a reliable ELISA screening to determine the presence of shed PrP (PrP226*) in blood donor plasma samples using mAb V5B2 as a primary antibody and using mAb E12/2 as a detection antibody. The sensitivity of the test was determined, and then 484 blood donor plasma samples were tested. After screening, we came to the conclusion that due to the presence of heterophile antibodies (HA) we received false positive test results. The presence of HA in the sample was confirmed using commercially available antibodies. We then tried to extract HA from the sample using various methods. Prionski protein PrP226* biomarker določanje ELISA heterofilna protitelesa Prion protein PrP226* biomarker detection ELISA heterophilic antibodies true false false [R. Pucko] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2020-09-19 07:15:04 2020-09-19 07:15:18 2022-09-01 03:58:41 0000-00-00 00:00:00 2020 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 606:61:57.083.3:602.44:547.963.1(043.2) 181101 32311555 10671.pdf 10671.pdf 1 8574266CF2CF6C688E98E18C9654F907 591a3b22b9a879382a6e77db7dcb65c16b0d3fc38cf83837c5beb5f1db7bafda 128c4f44-a1ba-11eb-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=135167 Biotehniška fakulteta 0 0 0