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Izbira primernih monoklonskih protiteles in optimizacija imunohistokemijske reakcije za dokazovanje patološkega prionskega proteina v možganih bolnikov s sporadicno obliko Creutzfeldt - Jakobove bolezni
Avtor: Senta Frol, Martin Hren
Mentor: doc.dr. Mara Popovic, dr.med
Somentor: doc.dr. Vladka Curin-Šerbec, univ.dipl.kem.
Izhodišce: Zanesljiva diagnoza prionskih bolezni sloni na imunohistokemiji ob uporabi protiteles proti prionskemu proteinu (PrP), ki ne razlikujejo med celicno obliko PrP (PrPC) in patološko izoobliko (PrPSc). Zato ta metoda zahteva mocne predobdelave za odstranjevanje PrPC in razkrivanje epitopa na PrPSc v parafinskih rezinah umrlih za prionsko boleznijo.
Namen: Namen naše naloge je testirati za PrPSc domnevno specificna monoklonska protitelesa (MPt-a) V5B2, K4B3, A4/5 in E5/9, ki so jih pripravili na Zavodu RS za transfuzijo krvi, jih primerjati s kontrolnimi, komercialno dostopnimi MPt-i 3F4 in 6H4, ter poiskati enostavnejšo predobdelavo za razkrivanje epitopa na PrPSc.
Hipoteza: Domnevali smo, da so vsa štiri naša MPt-a specificna za PrPSc, da se razlikujejo po obcutljivosti in da bo mogoce vsaj eno izmed njih uporabljati za zanesljivo diagnostiko prionskih bolezni brez mocnih predobdelav možganskega tkiva.
Metode: Parafinske histološke rezine vzorcev možgan štirih umrlih za sporadicno Creutzfeldt-Jakobovo boleznijo (sCJB) in dveh umrlih primerljive starosti, ki nista imela CJB, smo obdelali z imunohistokemijsko metodo streptavidin-biotin kompleks ob uporabi naših MPt-s, kontrolnih MPt-s in štirih predobdelav. Imunohistokemijsko reakcijo (IHR) med našimi MPt-i in PrPSc smo primerjali z IHR med kontrolnima MPt-oma (3F4 in 6H4) in PrPSc z ozirom na jakost signala IHR in število pozitivnih reakcij. Ista MPt-a smo uporabili tudi na zaledenelih rezinah vzorcev dveh ne-CJB možgan brez kakršnekoli obdelave. Rezultate smo statisticno obdelali s t-testom.
Rezultati: V nalogi smo pokazali, da so vsa štiri testirana MPt-a specificna za PrPSc, saj za razliko od dveh kontrolnih MPt-s niso prikazala PrPC v zaledenelih rezinah svežih ne-CJB možgan. Prav tako smo pokazali, da se z mocnejšimi predobdelavami tkiva IHR izboljša, tako v jakosti kot tudi v številu pozitivnih reakcij (p<0,05; enosmerni t-test). Naše MPt V5B2 je bolj obcutljivo od kontrolnega MPt-a 3F4 (p=0,03; enosmerni t-test).
Zakljucki: Z omenjenimi rezultati smo potrdili našo hipotezo o specificnosti vseh štirih naših MPt-s za PrPSc. Prav tako smo pokazali, da se ta MPt-a med seboj razlikujejo po obcutljivosti in da je za diagnostiko sCJB najbolj primerno MPt V5B2, primernejše tudi od dveh kontrolnih MPt-s. Domnevamo, da je MPt V5B2 zaradi svoje specificnosti in obcutljivosti, primeren za izdelavo testov za dokazovanje PrPSc v zaledenelih rezinah potencialno okuženih možgan ali pa v telesnih tekocinah.
[Abstract / English version]
Selection of suitable monoclonal antibodies and optimisation of immunohistochemical method for detection a pathological prion protein in brain of patients with a sporadic form Creutzfeldt -Jakob disease
Author: Senta Frol, Martin Hren
Mentor: doc.dr. Mara Popovic, dr.med
Co-mentor: doc.dr. Vladka Curin-Šerbec, univ.dipl.kem.
Background: A reliable diagnosis of the prion diseases is based on immunohistochemistry by using the antibodies against prion protein (PrP) which, unfortunately, do not make any distinction between the normal PrP (PrPC) and pathological isoform (PrPSc). Consequently this method requires intensive pre-treatments to remove PrPC and to retrieve the epitop of PrPSc in paraffin sections of brain with prion disease.
Aim: The aim of this study is to test allegedly PrPSc specific monoclonal antibodies (MAb-s) V5B2, K4B3, A4/5 and E5/9 which were prepared by the Centre for Blood Transfusion of Republic of Slovenia, to compare them with commercially accessible MAb-s 3F4 and 6H4 and to design simple antigen retrieval method to label PrPSc in immunohistochemistry.
Hypothesis: It was assumed that all our four MAb-s are specific for PrPSc , that they differ in sensitivity and at least one of them presumably may serve for reliable diagnostic of prion diseases without requiring intensive pretreatment of brain tissues.
Methods: The slides of paraffin embedding brain samples of four persons died for sporadic Creutzfeldt _Jakob disease (sCJD) and of two deceased patients of comparable age who had not had the CJD were treated with immuno histochemical method streptavidin -biotin complex with using our MAb-s, control MAb-s and four pretreatments. Immunohistochemical reaction (IHR) of our MAb-s was compared with IHR of control MAb-s (3F4 and 6H4) regarding intensity of IHR and the number of positive reactions. The same MAb-s were tested on the frozen sections of two brains not affected by CJD without any pre-treatment. The results were statistically processed with t-test.
Results: The results of the task showed that all four tested MAb-s were specific for PrPSc since, unlike the two control MAb-s, did not react with PrPC in the frozen sections of non-CJD brain. An improvement in IHR at more intense pretreatments of tissues in terms of intensity and the number of positive reactions (p<0.05; one way t-test) was proved as well. Our MAb V5B2 is more sensitive than the control MAb 3F4 (p=0,03; one way t-test).
Conclusions: The obtained results confirmed the hypothesis on specificity of our MAb-s for PrPSc. Further we found that MAb-s are different in terms of sensitivity and that MAb V5B2 is best suitable for the diagnostic of CJD, more suitable even than the two control MAb-s. Due to its specificity and sensitivity MAb V5B2 is assumed to be suitable for preparing the tests able to detect native PrPSc in frozen sections of potentially infected brains or in body fluids of the patients with prion diseases.