The research centered on the development of an antibody-based optical biosensor. The enzyme lactate dehydrogenase was chosen as the analyte, as it is a clinically important enzyme and is used as an indicator of various disease states such as heart disease. Polyclonal antibodies were raised in rabbits. These antibodies were purified, characterised and labelled with horse radish peroxidase and fluorescein. Enzyme-linked and fluorescent-linked immunoassays were developed using the HRP-labelled and FITClabelled antibodies, respectively.
Antibodies specific for chicken heart lactate dehydrogenase (CHLDH), were immobilised on the exposed core of optical fibres. The immobilisation procedure was optimised to ensure reproducibility of immunoglobulin immobilisation. An optical immunosensor, based on the evanescent wave technique, was developed and optimised. The sensor was shown to be specific for CHLDH and sensitive to varying concentrations of CHLDH. A comparison was made between one-step and two-step assay systems using the evanescent wave immunosensor and the BIAcore (commercially available biosensor based on surface plasmon resonance). The sensors were also compared in an experiment to determine the effect of high levels of fluorophore labelling on antibody affinity. Anti-human H4LDH (LDHj isoenzyme) antibodies were also raised and tested in an ELISA system using human serum samples. The isoenzyme profiles of these serum samples were also prepared to see if elevated LDHX (H4LDH) levels could be used as an indicator of myocardial infarction. The specificity of the antibodies was checked using immunoblotting techniques.
A minituarised colourimetric assay for total LDH was developed and validated using patient samples.