Ultra-sensitive Immuno-qPCR based Avian Influenza H5 Detection using Aptamers Selected from Multiple SELEX libraries
Date Issued
2015
Date
2015
Author(s)
Wang, Ting-Hsien
Abstract
Aptamers, single-stranded nucleotides selected through Systematic Evolution of Ligands by EXponential enrichment (SELEX) that recognize its target protein specifically, are now considered promising in rapid diagnosis of influenza virus. However, identifying high affinity and specificity aptamer sequences in short time at low sample consumption have always been the bottleneck of SELEX works. In this thesis, an efficient micro-beads based SELEX was demonstrated with 3 distinctly primered ssDNA libraries in screening of H5 ultra-sensing aptamers by using recombinant H5 hemagglutinin (HA) as the selecting target. The result indicated that the micro-beads based SELEX was capable of obtaining ssDNA pools with low dissociation constant (~nM) and high convergence (>90%) in very early round of selection (<4). Among the selected aptamer candidates, ISP14 possessed outstanding affinity (45 pM) to the HA and was also validated being able to perform immuno-qPCR based HA detection with nano-molar ranged LOD in presence of interfering HSA, which showed great potential in clinical influenza virus detection. Furthermore, the in silico docking result provided evidences that selected aptamers bind to different epitopes on HA, implying chance of finding bivalent aptamers with avidity effect, which may dramatically improve the sensitivity of influenza virus detection.
Subjects
aptamer
micro-beads based SELEX
H5 hemagglutinin
immuno-qPCR
pico-molar affinity
avidity
Type
thesis
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