The Development of ELISA and SPR-Based Immunoassays for the Detection of Heat Shock Proteins.
Gong, B. (2009). The Development of ELISA and SPR-Based Immunoassays for the Detection of Heat Shock Proteins. (Thesis, Master of Science (MSc)). The University of Waikato, Hamilton, New Zealand. Retrieved from http://hdl.handle.net/10289/3275
Permanent Research Commons link: http://hdl.handle.net/10289/3275
Heat shock proteins (Hsps) are highly conserved molecules in all eukaryotes and prokaryotes. They are named on the basis of their molecular weight and their synthesis is up-regulated by stress conditions such as inflammation, oxidative stress and exposure to high temperature. They function by assisting the folding of newly synthesized proteins and thus prevent aggregation or damage to other cellular components. Beside the role in intracellular protein folding, heat shock proteins can particularly act as intercellular signals with a wide variety of biological effects, such as to stimulate cells to produce proinflammatory cytokines, like TNFα, and other proteins involved in immunity and inflammation, or they are also thought to be involved in the pathogenesis of a wide range of diseases such as diabetes mellitus and cardiovascular disease. Thus, screening for aberrant expression of this protein could be an easy and useful tool to detect at risk individuals for developing and those more susceptible towards developing these diseases. Currently, the Enzyme-Linked ImmunoSorbent Assay (ELISA) is the main immunoassay method used for the measurement of heat shock proteins levels in both clinical and research laboratories. It is relatively more rapid and sensitive than RadioImmunoassay (RIA), and most importantly, it is safer because enzymes are used as labels instead of harmful radioactive substances. However, a newly developed biosensor method, the Surface Plasmon Resonance (SPR), offers a number of advantageous over ELISA. For example, it is much more simple and rapid because a lot of steps can be set up automatically and no labels are required for SPR immunoassays. In this study, a comparison of different types of ELISA assays was made. The results showed that the Sandwich format of ELISA is much more sensitive than Indirect ELISA for detection of the concentrations of Heat Shock Protein 70 (Hsp70), and this sensitivity can be further improved by applying an Avidin-Biotin system together with Sandwich ELISA under certain conditions. To develop the SPR protocol for the detection of heat shock protein concentrations, two Hsp70 binding curves using different assay formats, the Sandwich SPR immunoassay and the Competitive SPR immunoassay, were set up by using CM 5 sensor chip. Another sensor chip, the mixed Self-Assembled Monolayer (mSAM), was also examined using Sandwich SPR format. No subsequent experimental steps were carried on for SPR studies since the regeneration conditions of these tests for both sensor chips need to be well studied. Thus, this study suggests that in order to set up a SPR immunoassay protocol that is a better choice for the detection of heat shock protein levels in complex matrixes than ELISA, experimental conditions, such as the choice of regeneration buffer and the duration of regeneration cycle, need to be well optimized.
The University of Waikato
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