Designing high sensitivity ELISAs through antibody orientation

Submission note: Submitted in fulfilment of the requirement of the Degree of Doctor of Philosophy to the Centre for Materials and Surface Science, Department of Chemistry and Physics, School of Molecular Sciences, La Trobe University, Bundoora, in conjunction with Manufacturing, Commonwealth Scientific and Industrial Research Organization (CSIRO), Melbourne.

Thesis with publications.

Enzyme-linked immunosorbent assays (ELISAs) utilize immobilized capture antibodies at solid-phase interfaces with enzymatic reporter molecules for antigen detection. ELISAs are employed globally for the detection and quantification of economically, medically, environmentally and industrially important antigens. In this work, a chromium complex containing the hexahydroxychromium (III) anion ([Cr(OH)6] 3- ) was used as a post-production, wet chemical modification to treat commercially available microtitre plates, for improved antibody immobilization and subsequent use in ELISA. The chromium modified plates improve the density and orientation of the capture antibody by binding its Fc portion, resulting in increased signal intensity, lower antigen detection limits, and increased speed of ELISAs. Production of the metal complex via various routes was explored using high-throughput methodology. The metal complex modification was then exemplified using a range of microtitre plates and sandwich ELISA systems. A diethylene glycol dimethyl ether plasma polymer thin film (DGpp) was then developed as a coating for microtitre well plates that provided a substrate for chromium binding. The resultant film, DGpp+Cr, offered a further ELISA improvement over previous methods with the enhanced signal due to correct orientation of captured antibody rather than a higher density of antibody binding. Time-of-flight secondary-ion mass spectrometry (ToF-SIMS) methods and multivariate analysis techniques, principal component analysis (PCA) and artificial neural networks (ANNs), were developed to probe antibody orientation on solid surfaces. These were use to directly demonstrate improved antibody orientation on DGpp+Cr over DGpp alone. ToF-SIMS, PCA and ANN were also employed to investigate classification of an antibody from its proteolytic fragments immobilized on silicon substrates. These methods were utilized to investigate antibody denaturation at the interface after ultrahigh vacuum storage, resulting in a "denaturation ratio" that is substrate and primary-ion independent. This research will be instrumental in improving not only immunoassay capabilities, but also mass spectral and multivariate analysis techniques employed to investigate complex biological systems.