Novel compounds for immunoassay of small biomolecules
Mitchell, J. S. (2005). Novel compounds for immunoassay of small biomolecules (Thesis, Doctor of Philosophy (PhD)). The University of Waikato, Hamilton, New Zealand. Retrieved from https://hdl.handle.net/10289/13252
Permanent Research Commons link: https://hdl.handle.net/10289/13252
A series of progesterone-4-ovalbumin conjugates with different length intermediate linkers were applied as coating antigens in an enzyme-linked immunosorbent assay (ELISA) format to determine antibody-binding performance and assay parameters in cow whole milk. The use of an 18-atom linker gave higher binding than 4- or 11-atom linkers but no further increase was seen with increasing linker length. An ELISA constructed with the 18-atom linker conjugate gave a detection limit of 0.089 ng/mL progesterone and correlated well to an established radioimmunoassay procedure (r = 0.94). The assay has the advantages of a wide linear range (0.1 to 100 ng/mL) enabling full profiling of bovine estrous cycles, use of whole milk directly without extraction or predilution, and employing more easily purified protein conjugates as coating antigens compared with commercial progesterone-enzyme conjugates. Thioether-bridged derivatives of 17~-estradiol and estrone were produced with attachment at the 4-position of the aromatic A-ring using aromatic substitution of the corresponding bromoestrogens. These thioether derivatives were then modified further to attach oligoethylene glycol or polyethylene / polypropylene glycol chains of varying length (n = 3 and n = 15-19). The estradiol-oligoethylene glycol derivative was immobilised in situ in a surface plasmon resonance (SPR) biosensor and its antibody binding compared to conjugations through the steroid 2-position by Mannich reaction and the 3-position by hemisuccinate conjugation. 2-position conjugation gave 19% more binding than 4-position and 3-position hemisuccinate gave negligible binding to the antibody raised to a 6-position conjugate. The chip surfaces were then used to construct SPR assays of estradiol with and without secondary antibody signal enhancement and gave limit of detection (LOO) as low as 25 pg/mL. Thioether-bridged derivatives of the catecholamines dopamine, norepinephrine and epinephrine were synthesised by electrolysis of the parent catecholamine to produce the corresponding o-quinone and then simply adding a thiol of three or 11-carbons in length with a terminal carboxylic acid function. A convenient cationic exchange method was developed for purification of the dopamine and nor-epinephrine products, whilst the epinephrine derivatives could be purified on normal-phase silica. These reactions and separations were followed by reversed-phase HPLC. The dopamine propionate derivative was applied as coating antigen by in situ surface assembly with two different linkers and compared to conjugation through the amine. The antibody raised to a glutaraldehyde-linked conjugate showed highest binding to the amine-conjugated surface. New surface plasmon resonance biosensor formats were developed using gold nanoparticles to amplify the binding signal of progesterone / antibody binding interactions. A very stable sensor surface with progesterone immobilised via an oligoethylene glycol chain was produced capable of withstanding more than 1100 binding and regeneration cycles without appreciable loss of binding capacity. Gold labelling of the monoclonal antibody was achieved via a biotin/streptavidin couple in both a preincubation of biotinylated antibody with streptavidin gold and a sequential addition of antibody and then gold label. Preincubation gave no signal enhancement but lowered the detection limit of the assay to 143 pg/mL compared with previous reports which had detection limited to l ng/mL. Sequential labelling resulted in a two-fold enhancement of signal and the detection limit was reduced to 23.l pg/mL. Labelling with a secondary antibody alone produced signal enhancements of eight-fold and when secondary antibody was conjugated to colloidal gold it produced enhancements of 13-fold. The secondary antibody-gold labelling gave LOO of 8.6 pg/mL. There was no difference in enhancement between 10 and 20 nm colloid for the biotin/ streptavidin couple and colloid size did not affect enhancement for the secondary antibody-mediated format. This study is the first successful development of nanoparticle signal-enhanced immunoassay for small molecules using SPR and demonstrates how assay performance may be improved to levels comparable to or better than existing ELISA.
The University of Waikato
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