magistrsko delo
Samuel Žvanut (Author), Tom Turk (Reviewer), Metka Lenassi (Mentor)

Abstract

Kljub nekaterim testom za detekcijo rezervoarjev virusa humane imunske pomanjkljivosti tipa 1 (HIV-1), sposobnih ponovne infekcije, manjkajo metode, ki bi ovrednotile obsežnost translacijsko sposobnih in posledično biološko aktivnih rezervoarjev HIV-1. Nef se dokazano sprošča iz rezervoarjev HIV-1 v telesne tekočine in ima funkcijo v patogenezi HIV-1. V nalogi smo optimizirali encimskoimunski test (ELISA) za detekcijo virusnega proteina Nef. ELISA smo izvedli na osnovi nanotelesa sdAb19 proti Nef, ki smo ga biotinilirali in vezali na nevtravidinsko podlago. V pufru smo uspeli detektirati rekombinantni Nef (rNef) pri meji ozadja 4,25 ng/ml in meji detekcije 6,79 ng/ml, kar sovpada s fiziološkimi koncentracijami Nef v plazmi okuženih. Razpon koncentracij, v katerem je ELISA na osnovi sdAb19 kvantitativna, je med 10 ng/ml in 400 ng/ml. Pri detekciji rNef, ki smo ga raztopili v človeškem serumu, smo opazili visoko mejo ozadja, detekcija rNef pa je bila neuspešna. Ob uspešni optimizaciji testa v kompleksnih bioloških tekočinah bi s pristopom, ki smo ga opisali v nalogi, lahko relativno učinkovito ovrednotili obsežnost aktivnih rezervoarjev, vpletenih v patogenezo HIV-1.

Keywords

Nef;encimskoimunski test;nanotelo sdAb19;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL BF - Biotechnical Faculty
Publisher: [S. Žvanut]
UDC: 577:61(043.2)
COBISS: 164359427 Link will open in a new window
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Downloads: 2
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Other data

Secondary language: English
Secondary title: Optimization of nanobody-based immunoassay for detection of viral protein Nef in biological samples
Secondary abstract: Despite some tests for detecting reservoirs of human immunodeficiency virus type 1 (HIV-1) capable of causing reinfection, there is a lack of methods to assess the extent of translationally competent and consequently biologically active HIV-1 reservoirs. In this work, we optimized an enzyme-linked immunosorbent assay (ELISA) for detecting the viral protein Nef. Nef is released from HIV-1 reservoirs, is present in various biological fluids, and contributes to HIV-1 pathogenicity. The optimized ELISA utilized biotinylated anti-Nef nanobody sdAb19, immobilized on the neutravidin-coated surface. Recombinant Nef (rNef) was detected in a buffer with a limit of the blank at 4.25 ng/ml and a limit of detection at 6.79 ng/ml. These values align with physiological Nef concentrations in the plasma of infected individuals. The quantitative range of the sdAb19-based ELISA spans from 10 ng/ml to 400 ng/ml. Notably, we observed a high limit of the blank when detecting rNef diluted in human serum. Through further optimization of the assay in complex biological fluids, our method could prove efficient in evaluating the size of pathologically relevant active HIV-1 reservoirs.
Secondary keywords: Nef;enzyme-linked immunosorbent assay;nanobody sdAb19;
Type (COBISS): Master's thesis/paper
Study programme: 0
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Biotehniška fak., Oddelek za biologijo, Magistrsko delo magistrskega študijskega programa 2. stopnje Molekulske in funkcionalne biologije
Pages: 1 spletni vir (1 datoteka PDF (IX, 54 str.)
ID: 19929381