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Title: Multiplexed nanoparticle-based immunoassays
Author: Murray, K. A.
Awarding Body: Nottingham Trent University
Current Institution: Nottingham Trent University
Date of Award: 2010
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Multiplexed immunoassays have been explored using the fluorescent and luminescent properties of fluorophores and nanoparticles. Epi-fluorescence microscopy, confocal laser scanning microscopy and programmable array microscopy were used to detect signals from mixtures of conventional organic fluorophores, quantum dots and silica nanoparticles doped with europium, samarium and terbium in single-welled multiplexed immunosorbent assays. Spectral unmixing was investigated using mixtures of fluorophores and cadmium selenide quantum dots. Mixtures of up to four dyes were separated quantitatively using least squares minimisation, with relative standard error ranging from 0.5 to 13 %. Silica nanoparticles doped with luminescent lanthanides were synthesised and used in a model immunoassay system for simultaneous, single-welled detection of human and mouse IgGs. The results indicated the lanthanides are well suited to multiplexed assays, mainly because of their atomic line emission bands. Analytes in a mixture could be quantified with < 5 % error. The multiplexed assay developed was applied to the detection of anti-dengue IgM and IgG in mouse sera, to differentiate primary and secondary dengue infection. The assay traced the kinetics of antibody production for both IgM and IgG with an IgM/IgG ratio of 1. The fluorescencebased methods compared favourably with ELISA results (r2 ≥ 0.8), with results using conventional fluorophores showing the best correlation with ELISA (r2 > 0.98). Differentiation of serotype specific IgGs was also explored but was complicated due to cross reactivity of the antibodies. A model to differentiate cross reactive dengue antigens was applied to the data through fitting parameters of the five parameter logistic equation to the intensity obtained for an assay. Results indicated the family of dengue antigens are too closely related to be distinguished by the method. The model was however successful in differentiating a partially cross reactive system (p > 0.95).
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available