Lara Smole (Author), Saša Čučnik (Mentor)

Abstract

Vnetje je osrednji proces kroničnih vnetnih bolezni, kjer pomembno vlogo v patogenezi igra citokin, dejavnik tumorske nekroze-α (TNF-α). Protivnetna biološka zdravila so večinoma terapevtska monoklonska protitelesa, med katere uvrščamo zaviralce TNF-α, ki nevtralizirajo prosti citokin in preprečijo njegovo vezavo na receptor. Infliksimab (IFX) je primer zaviralca TNF-α, ki je himerno, mišje-človeško monoklonsko protitelo. Sprva so biološka zdravila veljala za zelo varna in učinkovita, sčasoma pa so odkrili neželene učinke povezane z njimi, predvsem pojav primarne rezistence in sekundarne odpovedi. Pomembno je stalno spremljanje koncentracij biološke učinkovine pri posameznem bolniku, saj lahko le tako ustrezno prilagodimo terapijo. Najpogosteje uporabljena tehnika za detekcijo zaviralcev TNF-α je encimsko-imunska metoda na trdnem nosilcu (ELISA). Namen magistrske naloge je postavitev in ovrednotenje hišne ELISA za detekcijo IFX z uporabo dveh različnih detekcijskih protiteles in primerjava rezultatov dobljenih z analiznim kompletom apDia. Dobro postavljena in ovrednotena metoda je osnovni pogoj za nadaljnje vrednotenje rezultatov, zato smo posamezno metodo najprej ovrednotili. Pri izračunu znotraj-analizne in med-analizne natančnosti obeh hišnih IFX-ELISA je bil koeficient variabilnosti (KV%) pod mejo 20 %. Točnost je bila pri uporabi sekundarnega protitelesa, usmerjenega proti IFX in konjugiranega s hrenovo peroksidazo v mejah 80 – 120 %, pri uporabi proti človeškemu IgG usmerjenega sekundarnega protitelesa konjugiranega z alkalno fosfatazo pa 120,1 ± 11,2 %. Pri vrednotenju linearnosti smo dobili vse rezultate v mejah 80 – 120 %. Različica hišne metode z uporabo sekundarnega protitelesa, usmerjenega proti IFX in konjugiranega s hrenovo peroksidazo je analizno specifična. Pri primerjavi rezultatov hišne metode z analiznim kompletom apDia smo dosegli zelo dobro ponovljivost (r med 0,90 in 0,99 pri p<0,001). Povprečno smo dobili pri obeh različicah hišne metode nekoliko višje koncentracije kot pri apDia IFX-ELISA. Pri uporabi anti-IFX sekundarnega protitelesa konjugiranega s hrenovo peroksidazo smo dosegli 100 % pravilnost uvrščanja negativnih bolnikov. Glede na rezultate lahko zaključimo, da sta obe različici hišne IFX-ELISA primerljivi z rezultati analiznega kompleta apDia. Obe sta uporabni za detekcijo IFX, z vidika analizne specifičnosti in pravilnosti uvrščanja negativnih rezultatov pa je boljša različica hišne IFX-4 ELISA, ki uporablja specifično, anti-IFX sekundarno protitelo konjugirano s hrenovo peroksidazo.

Keywords

vnetne bolezni;tumorska nekroza-alfa;infliksimab;encimsko imunska metoda;trdni nosilec;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL FFA - Faculty of Pharmacy
Publisher: [L. Smole]
UDC: 61:616-002(043.3)
COBISS: 4462449 Link will open in a new window
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Downloads: 90
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Other data

Secondary language: English
Secondary title: Development and validation of an enzyme-linked immunosorbent assay to detect infliximab
Secondary abstract: Inflammation is the main process of chronic inflammatory diseases where cytokine, tumor necrosis factor-α (TNF-α) plays an important role in pathogenesis. Anti-inflammatory biological drugs are mostly therapeutic monoclonal antibodies. This group includes TNF-α inhibitors that neutralize free cytokine and prevent its binding to the receptor. Infliximab (IFX) is an example of TNF-α inhibitor that is a chimeric, mouse/human monoclonal antibody. At first, biological drugs were believed to be very safe and effective, but over time, side effects were shown- for example primary and secondary response failure. It is important to monitor the levels of biological drug constantly, as this is the only way to adjust the therapy for the individual patient. Enzyme-linked immunosorbent assay (ELISA) is the most commonly used technique for the detection of TNF-α inhibitors. The purpose of thesis is to develop and evaluate an in-house IFX-ELISA, using two different detection antibodies and to compare in-house ELISAs with the apDia IFX-ELISA kit. A well-established and evaluated method is the basis for further assessment of the results, so we first evaluated each method. In case of intra-assay and inter-assay precision the coefficient of variation (CV%) was lower than 20% for both variants of in-house IFX-ELISA. In case of anti-IFX secondary antibody conjugated with horseradish peroxidase used in in-house ELISA recovery was between 80 - 120%. When we used secondary antibody directed against human IgG conjugated with alkaline phosphatase recovery was 120,1% ± 11,2%. For linearity, the results we obtained were all within the limits of 80 - 120%. The variant of in-house IFX-ELISA in which we used anti-IFX secondary antibody conjugated with horseradish peroxidase is analytically specific. Comparison with apDia IFX-ELISA kit yielded very good repeatability (r between 0.90 and 0.99, p<0,001). In both versions of the in-house method, on average we obtained a slightly higher concentration than apDia IFX-ELISA. When using the anti-IFX secondary antibody conjugated with horseradish peroxidase 100% accuracy of classification of negative patients was achieved. In conclusion, both variants of an in-house IFX-ELISA give comparable results to apDia IFX-ELISA kit. Both are useful for detection of IFX, but in terms of analytical specificity and accuracy of negative results classification, the variant with specific anti-IFX secondary antibody conjugated with horseradish peroxidase is better.
Secondary keywords: Citokini;
Type (COBISS): Master's thesis/paper
Thesis comment: Univ. Ljubljana, Fak. za farmacijo
Pages: 54 f.
ID: 12040940